Prodrugs activated by plasmin and their use in cancer chemotherapy

ABSTRACT

The product of the invention is a modified form of a therapeutic agent and comprises a therapeutic agent, an oligopeptide having a plasmin peptide substrate of 2-4 amino acids and mono- or di-peptide linkage, a stabilizing group and, optionally, a linker group. The prodrug is cleavable by plasmin. Also disclosed are methods of making and using the prodrug compounds.

TECHNICAL FIELD

The present invention relates to compounds useful as prodrugs that areactivated by tumor-secreted enzymes. More particularly, the inventionrelates to prodrugs having a novel mono- or di-peptide linkage and aplasmin peptide substrate of 2-4 amino acids. Such prodrugs may be usedfor treatment of disease, especially in cancer chemotherapy.

BACKGROUND

Many therapeutic agents, such as anthracyclines and vinca alkaloids, areespecially effective in cancer chemotherapy. However, these agents oftenexhibit acute toxicity iii vivo, especially bone marrow and mucosaltoxicity, as well as a chronic cardiac toxicity in the case of theanthracyclines and chronic neurological toxicity in the case of thevinca alkaloids. Similarly, methotrexate may be used for the treatmentof inflammatory reactions, such as rheumatic diseases, but its hightoxicity limits its applications. Development of more and safer specificantitumor agents is desirable for greater effectiveness against tumorcells and a decrease in the number and severity of the side effects ofthese products (toxicity, destruction of non-tumor cells, etc.).Development of more specific anti-inflammatory agents is also desirable.

The search for more selective anticancer agents has been extremelyactive for many decades, the dose limiting toxicities (i.e. theundesirable activity of the anticancer agents on normal tissues) beingone of the major causes of failures in cancer therapy. Accordingly, thegoal has been to improve the specificity of anti-tumor agents forincreased effectiveness against tumor cells, while decreasing adverseside effects, such as toxicity and the destruction of non-tumor cells.

The focus of research has been on the development of new therapeuticagents which are in the form of prodrugs, compounds that are capable ofbeing converted to drugs (active therapeutic compounds) in vivo bycertain chemical or enzymatic modifications of their structure. Forpurposes of reducing toxicity, this conversion is preferably confined tothe site of action or target tissue rather than the circulatory systemor non-target tissue. Prodrugs are often characterized by a lowstability in blood and serum, due to the presence of enzymes thatdegrade or activate the prodrugs before the prodrugs reach the desiredsites within the patient's body. A desirable class of prodrugs thataddresses such problems has been disclosed in Trouet, et al., U.S. Pat.No. 5,962,216 and in Lobl, et al., PCT International Publication No. WO00/33888, both of which are incorporated herein by reference.

Tripeptide derivatives of anticancer agents such as daunorubicin (“DNR”)and doxorubicin (“DOX”), to be used as prodrugs were studied byChakravarty, et al., J. Med. Chem. 26:633-638, 1983 (A); Chakravarty, etal., J. Med. Chem. 26:638-644, 1983 (B); and Balajthy, et al., J. Med.Chem. 35:3344-3349, 1992. However, none of these approaches has beenshown to be successful.

Other work in this area includes Monsigny, et al., FEBS Letters119(1):181-186, 1980 (DNR); Baurain, et al., U.S. Pat. No. 4,296,105(DNR); Baurain, et al., J. Med. Chem. 23:1171-1174, 1980 (DNR);Masquelier, et al., J. Med. Chem. 23:1166-1170, 1980 (DNR); and deGroot, et al., J. Med. Chem. 42:5277-5283, 1999 (DNR and DOX), which alldescribe prodrugs comprising a carrier linked to the drug via a peptidearm. Typically, these references describe a peptide arm, linked via itsfree carboxyl function to the free amine function of derivatives ofanthracyclines such as DNR. In addition, the arm of these prodrugs canbe linked via its free amine function to a carrier consisting of amacromolecule (protein such as BSA, immunoglobulins, etc.) which permitsthe selective endocytosis of the prodrug by target cells.

However, in spite of the advances in the art, there continues to be aneed for the development of useful prodrug compounds and methods ofmaking such prodrugs. Prodrugs that display a high specificity ofaction, reduced toxicity, and improved stability in blood relative toknown prodrugs of similar structure are particularly desirable. Theinstant invention addresses those needs.

SUMMARY OF THE INVENTION

One aspect of the invention relates to compounds that are prodrug formsof therapeutic agents with a mono- or di-peptide linkage and a plasminpeptide substrate of 2-4 amino acids (i.e., a dipeptide, tripeptide ortetrapeptide substrate), which in turn, is linked to a stabilizinggroup. In essence, the prodrug comprises a plasmin-recognizable peptidicsequence linked to one or two aliphatic amino acids having a largelateral chain which aliphatic amino acid is further linked to the drug.These prodrugs display a high specificity of action, reduced toxicity,improved stability in the serum and blood, and exhibit minimal movementinto target cells unless activated by a target cell associated enzyme.

In one aspect of the invention, the compound comprises: (1) atherapeutic agent capable of entering a target cell; (2) an oligopeptidehaving the formula X-Y, where X is a plasmin peptide substrate of 2-4amino acids and Y is a peptide comprising 1-2 aliphatic amino acidshaving large lateral chains; (3) a stabilizing group; and (4)optionally, a linker group not cleavable by plasmin; wherein theoligopeptide is directly linked to the stabilizing group at a firstattachment site of the oligopeptide and the oligopeptide is directlylinked to the therapeutic agent or indirectly linked through the linkergroup to the therapeutic agent at a second attachment site of theoligopeptide; wherein the stabilizing group hinders cleavage of theoligopeptide by enzymes present in whole blood; and wherein the compoundis cleavable by plasmin. In anther aspect of the invention, the compoundis selectively cleavable by plasmin.

In another aspect of the invention, the oligopeptide has the formula(AA^(x))_(m)-(AA^(y))_(n) (SEQ ID NO:1) wherein: (AA^(x))_(m) is aplasmin substrate and each AA^(x) independently represents an aminoacid; each AA^(y) independently represents an aliphatic amino acidhaving a large lateral chain; m is an integer from 2-4; and n is aninteger from 1-2, said oligopeptide being cleavable by plasmin.

Another aspect of the invention pertains to a pharmaceutical compositioncomprising these prodrugs and optionally a pharmaceutically acceptablecarrier.

Yet another aspect of the invention pertains to compounds comprising amarker useful in the characterization of tumors, for example, diagnosis,progression of the tumor, and assay of the factors secreted by tumorcells. Another aspect of the invention relates to articles ofmanufacture for diagnosis or conducting assays comprising: (1) acompound comprising (a) a marker, (b) the oligopeptide described above,(c) a stabilizing group, and (d) optionally, a linker group notcleavable by plasmin; and (2) at least one reagent useful in thedetection of the marker.

Another aspect of the invention relates to methods of treating a medicalcondition by administering a prodrug of the invention to a patient in atherapeutically effective amount.

Yet another aspect of the invention pertains to a method for decreasingthe toxicity of a therapeutic agent wherein the therapeutic agent isintended for administration to a patient, the method comprising:covalently forming a prodrug by linking the oligopeptide described aboveto a stabilizing group at a first attachment site of the oligopeptideand directly or indirectly linking the therapeutic agent at a secondattachment site of the oligopeptide, whereby the prodrug provides fordecreased toxicity of the therapeutic agent when administered to thepatient.

DETAILED DESCRIPTION OF THE INVENTION

The invention describes prodrugs that (1) are sufficiently stable in thebloodstream and in the biological fluids, to achieve importantconcentrations at tumor level in its non-hydrolyzed form; (2) are unableto enter, as such, into either normal or tumoral cells; and (3) areactivated extracellularly by proteases secreted by tumor cells andhaving a hydrolysis rate considerably high to provide the liberation ofsufficient quantities of active agent capable of entering cells andreaching its intracellular targets. The prodrugs are activated byplasmin, an enzyme overproduced in the vicinity of a large number ofhuman tumor cells.

Before proceeding with the description of the invention, it may behelpful to set forth the abbreviations used herein.

Abbreviations ACN Acetonitrile B16—B16 B16—B16 melanoma cells BFS Bovinefetal serum Boc t-Butyloxycarbonyl BSA Bovine serum albumin Bz BenzylCbz Benzyloxycarbonyl CM Conditioned medium DBN 1,5 Diazabicyclo [4.3.0]non-5-ene DBO 1,4 Diazabicyclo [2.2.2] octane DBU 1,8-Diazabicyclo[5.4.0] undec-7-ene DCC N,N′Dicyclohexylcarbodiimide DCM DichloromethaneDIC N,N′-Diisopropylcarbodiimide DIPEA Diisopropylethylamine DMEMDulbecco's Modified Eagle Medium DMF Dimethylformamide DNR DaunorubicinDMSO Dimethylsulfoxide DOX Doxorubicin EDTA Ethylenediaminetetraaceticacid, tetrasodium salt EtOH Ethanol Et₂O Diethyl ether Fmoc9-Fluorenylmethyloxycarbonyl HATUO-(7-Azabenzotrazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate HBTU2-(1H-Benzotriazole-1-yl)1,1,3,3-tetramethyluronium- hexafluorophosphateHOBT N-Hydroxybenzotriazole HPLC High pressure liquid chromatographyKSCN Potassium isothiocyanate Me Methyl MeOH Methanol MeOSucc Methylhemisuccinate or methyl hemisuccinyl NaOAc Sodium acetate NMPN-methylpyrrolidone NMR Nuclear magnetic resonance OSu theN-hydroxysuccinimide ester PAM resin4-hydroxymethylphenylacetamidomethyl PBS Phosphate buffered saline:NaH₂PO₄ 2.8 mM, NaHPO₄ 7.2 mM, NaCl 150 mM, pH 7.4 PEG Polyethyleneglycol PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate Pyg Pyroglutamic acid RT Room temperature SPPSMerrifield solid phase peptide synthesis method Succ SuccinylAcid/Succinyl tBu tert-Butyl TCE trichloroethyl TEA Triethylamine TESbuffer Tris-HCl 50 mM, EDTA 50 mM in H₂O, pH 8.0 TFA Trifluroroaceticacid THF Tetrahydrofuran TLC Thin Layer Chromatography Tosp-Toluenesulfonyl

Prodrugs

The prodrug of the invention is a modified form of a therapeutic agentand comprises several portions, including: (1) a therapeutic agent; (2)an oligopeptide; (3) a stabilizing group; and (4) optionally, a linkergroup. Each of the portions of the prodrug are discussed in greaterdetail below. A typical orientation of a prodrug of the invention is asfollows:

(stabilizing group)-(oligopeptide)-(optional linker group)-(therapeuticagent) The stabilizing group is directly linked to the oligopeptide at afirst attachment site of the oligopeptide. The oligopeptide is directlyor indirectly linked to the therapeutic agent at a second attachmentsite of the oligopeptide. If the oligopeptide and the therapeutic agentare indirectly linked, then a linker group is present.

As used herein, the term “direct” linkage of two portions of the prodrugmeans a covalent bond exists between the two portions. The stabilizinggroup and the oligopeptide are directly linked via a covalent chemicalbond at a first attachment site of the oligopeptide. Similarly, theoligopeptide and the therapeutic agent can be directly linked by acovalent bond at a second attachment site of the oligopeptide.

As used herein, the term “indirect” linkage of two portions of theprodrug means each of the two portions is covalently bound to a linkergroup. Accordingly, in an alternative embodiment, the stabilizing groupand the oligopeptide are directly linked via a covalent chemical bond ata first attachment site of the oligopeptide and the oligopeptide and thetherapeutic agent are indirectly linked by a linker group at a secondattachment site of the oligopeptide.

The first attachment site of the oligopeptide is typically at theN-terminus and the second attachment site is typically at theC-terminus. However, in an alternative embodiment, the orientation ofthe prodrug may be reversed so that the stabilizing group is directlylinked to the C-terminus of the oligopeptide and the therapeutic agentis directly or indirectly linked to the N-terminus of the oligopeptide.Thus, in the alternative embodiment, the first attachment site may bethe C-terminus of the oligopeptide and the second attachment site of theoligopeptide may be the N-terminus of the oligopeptide. The alternativeembodiment of the invention functions in the same manner as does theprimary embodiment.

The oligopeptide portion of the prodrug of the invention comprises tworegions: a plasmin peptide substrate and a 1-2 aliphatic amino acidlinkage. In order for the prodrug to be effective, the prodrug typicallyundergoes in vivo modification and an active portion, i.e., atransport-competent portion, of the prodrug enters the target cell. Afirst cleavage within the oligopeptide portion of the prodrug may leavean active or transport-competent portion of the prodrug as one of thecleavage products. Alternatively, further cleavage by one or morepeptidases may be required to result in a portion of the prodrug that iscapable of entering the cell. The active portion of the prodrug has atleast the therapeutic agent and is that part of the prodrug that canenter the target cell to exert a therapeutic effect directly or uponfurther conversion within the target cell. Thus, the compound has anactive portion, and the active portion is more capable of entering thetarget cell after cleavage by plasmin, an enzyme associated with thetarget cell, than prior to cleavage by plasmin.

The structures of the stabilizing group and oligopeptide are selected tolimit clearance and metabolism of the prodrug by enzymes that may bepresent in blood or non-target tissue and are further selected to limittransport of the prodrug into the cells. The stabilizing group blocksdegradation of the prodrug and may act in providing preferable charge orother physical characteristics of the prodrug. The amino acid sequenceof the oligopeptide is designed to ensure specific cleavage by plasmin.

It is desirable to make a therapeutic agent, especially an antitumorand/or anti-inflammatory therapeutic agent, inactive by modification ofthe therapeutic agent to a prodrug form. According to the invention, thetarget cells are usually tumor cells or cells participating ininflammatory reactions, especially those associated with rheumaticdiseases, such as macrophages, neutrophils, and monocytes. Modificationof the therapeutic agent to a prodrug form also reduces some of the sideeffects of the therapeutic agents.

In the target cell, the therapeutic agent (optionally attached to one ortwo aliphatic amino acids and possibly also a linker group) acts eitherdirectly on its specific intracellular action site or, after amodification by intracellular proteases, kills the target cell or blocksits proliferation. Since normal cells do not activate plasminogen intoplasmin in vivo as is seen with tumor cells, the prodrugs of theinvention are maintained inactive and do not enter the normal cells ordo so to a relatively minor extent.

The prodrug is administered to the patient, carried through the bloodstream in a stable form, and when in the vicinity of a target cell, isacted upon by plasmin. Since the enzyme activity is only minimallypresent within the extracellular vicinity of normal cells, the prodrugis maintained and its active portion (including the therapeutic agent)gains entry into the normal cells only minimally. In the vicinity oftumor or other target cells, however, the presence of plasmin in thelocal environment allows for cleavage of the prodrug. Once the prodrugis cleaved extracellularly, the transport-competent or active portiongains entry into the target cell. Once within the target cell, it may befurther modified to provide therapeutic effect, such as by killing thetarget cell or blocking its proliferation. While a portion of theprodrug may occasionally gain access to, and possibly harm normal cells,the transport-competent portion of the drag is freed primarily in thevicinity of target cells. Thus, toxicity to normal cells is minimized.

This process is particularly useful for, and is designed for, targetcell destruction when the target tissue excretes an enzyme or otherfactor that is not secreted by normal cells. Here “normal cells” meansnon-target cells that would be encountered by the prodrug uponadministration of the prodrug in the manner appropriate for its intendeduse. Since normal cells liberate little or none of the target-cellenzyme(s) that are responsible for cleaving the bond that links theactive portion (including the therapeutic agent) of the prodrug from theremainder of the prodrug in vivo, the compound of the invention ismaintained inactive and does not enter the normal cells.

As described in greater detail below, the prodrugs of the invention arecompounds comprising:

(1) a therapeutic agent capable of entering a target cell;

(2) an oligopeptide having the formula X-Y, where X is a plasmin peptidesubstrate of 2-4 amino acids and Y is a peptide comprising 1-2 aliphaticamino acids having large lateral chains;

(3) a stabilizing group; and

(4) optionally, a linker group not cleavable by plasmin;

wherein the oligopeptide is directly linked to the stabilizing group ata first attachment site of the oligopeptide and the oligopeptide isdirectly linked to the therapeutic agent or indirectly linked throughthe linker group to the therapeutic agent at a second attachment site ofthe oligopeptide;

wherein the stabilizing group hinders cleavage of the oligopeptide byenzymes present in whole blood; and

wherein the compound is cleavable by plasmin.

As used herein, “cleavable by” means cleavable under physiologicalconditions.

The oligopeptide can also be described as: (AA^(x))_(m)-(AA^(y))_(n)(SEQ ID NO:1) wherein: (AA^(x))_(m) is a plasmin substrate and eachAA^(x) independently represents an amino acid; each AA^(y) independentlyrepresents an aliphatic amino acid having a large lateral chain; m is aninteger from 2-4; and n is an integer from 1-2.

Target Cell Associated Enzyme: Plasmin

The prodrugs of the invention are designed to take advantage ofpreferential activation through interaction with plasmin, an enzymeassociated with the target cell, at or near the site targeted within thebody of the patient. The involvement of proteases, such as theplasminogen activation system, in the cascade of events leading to themetastatic spread of cancer cells has been well documented. Based on theinvolvement of this system in malignancy, and on the concept andtechnology previously developed in the work on other prodrugs (Trouet,et al., U.S. Pat. No. 5,962,216), several attempts were made to developprodrugs of anticancer agents that could be activated by plasmin. Theanthracycline daunorubicin (“DNR”) was used as a model drug and severalpeptidic sequences were linked via their carboxyl end to the amino groupof the sugar moiety of the drug.

Based on previous experimental data, it was known that sequences of fouramino acid residues would allow the prodrug to be sufficiently stable inthe bloodstream and in the biological fluids. The peptide sequences usedwere selected according to the known substrate specificity of plasmin asdescribed in the literature. In particular, plasmin is a serine proteasewith trypsin-like specificity, cleaving lysine and arginine bonds. Inaddition, it is known that plasmin specifically recognizes some peptidicsequences (Lottenberg, et al., Meth. Enzymol. 80:341-361, 1981), andamong them D-Alanyl-Leucyl-Lysyl-M and D-Val-Leu-Lys-M, cleaving thepeptide bond between the Lysyl residue and the M marker (Smith, et al.,Thromb. Res. 17:393, 1980).

As far as plasmin specificity is concerned, several different sequencesare possible and have been previously described. Some of these othersequences have been tried for the generation of tripeptide derivativesof anticancer agents to be used as prodrugs, but with very littleactivity in vivo (Chakravarty, et al. (A), supra; Chakravarty, et al.(B), supra; and Balajthy, et al., supra. These prodrugs wereoligopeptidic derivatives of the respective anticancer agent, and weredesigned in such a way that plasmin action on these derivatives shouldhave regenerated the active compound at the tumor level. However, in thecase of anthracyclines (DOX, DNR, etc), these tripeptidic derivativesproved to be very poor substrates for plasmin (D-Val-Leu-Lys-DOXdescribed in Chakravarty, et al. (A), supra and Chakravarty, et al. (B),supra;). More recently, a self-immolative spacer was placed between thetripeptide D-Ala-Phe-Lys to provide prodrugs with relative selectivityto plasmin (de Groot, et al., supra).

In the instant invention, one or two aliphatic amino acids with largelateral side chains, preferably are intercalated between the plasminsubstrate sequences described in the art and an agent of interest. Inthe instant invention, plasmin substrate-Leu-DNR was evaluated, and theeffect of modifications of the first N-terminal residue on bloodstability was checked. A potentially interesting plasmin-activatedprodrug was then selected on the basis of its stability in whole bloodand of its degradation into Leu-DNR by purified plasmin as well as bymedia conditioned by a human breast cancer cell line (MCF-7/6). Thestructure of this prodrug was D-Ala-Leu-Lys-Leu-DNR (Compound I, DNR).An additional Leu residue was added to provide the prodrugD-Ala-Leu-Lys-Leu-Leu-DNR (Compound II, DNR).

It is understood that other amino acid sequences specifically recognizedby plasmin can also be coupled to therapeutic agents in a way to assurean efficient dipeptidyl-agent liberation.

A distinctive feature of the instant invention is the introduction ofthis single or double aliphatic residue between the specific peptidesequence cleaved off by plasmin and the therapeutic agent, creating aspacer linkage between the enzyme substrate and the drug. It is believedthat this spacer linkage facilitates the access of plasmin to the bondthat is cleaved. The aliphatic amino acid has a large lateral side chainis preferably a leucine residue. However, other aliphatic amino-acidshaving a large lateral chain are also suited for use in the invention.These include by way of example and not limitation, phenylalanine,isoleucine and valine residues.

While demonstrated for the drug DNR, the plasmin-sensitive peptidesequences of the invention (D-Ala-Leu-Lys-Leu- andD-Ala-Leu-Lys-Leu-Leu, for example) can be used not only to prepare DNRprodrugs, but also prodrugs of a series of other anticancer agents, aswell as of several chemo- or radiosensitizers. In addition, anotheraspect of the invention involves using this sequence, not only withdrugs that are currently used clinically, but also with agents thatcannot be used because of their prohibitive toxicity. Therefore, anotheraspect of the invention relates to a method of significantly reducingthe toxicity of an agent while conserving or enhancing its activity.

Stabilizing Group

An important portion of the prodrug is the stabilizing group, whichserves to protect the prodrug compound from cleavage in circulatingblood when it is administered to the patient and allows the prodrug toreach the vicinity of the target cell relatively intact. The stabilizinggroup protects the prodrug from cleavage by proteinases and peptidasespresent in blood, blood serum, and normal tissue. Particularly, in thepreferred embodiment, where the stabilizing group caps the N-terminus ofthe oligopeptide, and is therefore sometimes referred to as an N-cap orN-block, the stabilizing group serves to ward against peptidases towhich the prodrug may otherwise be susceptible.

Ideally, the stabilizing group is useful in the prodrug of the inventionif it serves to protect the prodrug from degradation, i.e., cleavage,when tested by storage of the prodrug compound in human blood at 37° C.for 2 hours and results in less than 20%, preferably less than 2%,cleavage of the prodrug by the enzymes present in the human blood underthe given assay conditions.

More particularly, the stabilizing group can be one of the following:

(1) a non-amino acid group, i.e., a group other than an amino acid; or

(2) an amino acid that is preferably either (i) anon-genetically-encoded amino acid having four or more carbons or (ii)aspartic acid or glutamic acid attached to the N-terminus of theoligopeptide at the β-carboxyl group of aspartic acid or the γ-carboxylgroup of glutamic acid; or

(3) a non-amino acid group covalently linked to an amino acid that ispreferably either (i) a non-genetically-encoded amino acid having fouror more carbons or (ii) aspartic acid or glutamic acid attached to theN-terminus of the oligopeptide at the β-carboxyl group of aspartic acidor the γ-carboxyl group of glutamic acid.

Examples of non-amino acid groups include, by way of example and notlimitation, dicarboxylic (or a higher order carboxylic) acid or apharmaceutically acceptable salt thereof. Since chemical radicals havingmore than two carboxylic acids are also acceptable as part of theprodrug, the end group having dicarboxylic (or higher order carboxylic)acids is an exemplary N-cap. The N-cap may thus be a monoamidederivative of a chemical radical containing two or more carboxylic acidswhere the amide is attached onto the amino terminus of the peptide andthe remaining carboxylic acids are free and uncoupled. For this purpose,the N-cap is preferably succinic acid, methyl hemisuccinate, adipicacid, glutaric acid, or phthalic acid, with adipic acid and succinicacid being most preferred. Other examples of useful N-caps in theprodrug compound of the invention include diglycolic acid, fumaric acid,naphthalene dicarboxylic acid, pyroglutamic acid, acetic acid, 1 or 2,naphthylcarboxylic acid, 1,8-naphthyl dicarboxylic acid, aconitic acid,carboxycinnamic acid, triazole dicarboxylic acid, gluconic acid,4-carboxyphenyl boronic acid, PEG and (PEG)_(n)-analogs, butanedisulfonic acid, and maleic acid. Other non-amino acid stabilizinggroups include BSA, Boc, Bz and Cbz, Tos, wheat germ agglutinin andsuccinylated wheat germ agglutinin and dextran.

Suitable amino acid stabilizing groups include, by way of illustrationand not limitation, βAla (linked via its carboxyl function to theoligopeptide), α-methyl-Ala, D-Val, D-Ala, D-Phe, D-Ile, D-Pro andpoly[N⁵-(2-hydroxyethyl)-L-glutamine], with β-Ala, α-methyl-Ala andD-Ala being preferred.

Suitable stabilizing groups that are made up of a non-amino acid groupcovalently linked to an amino acid, include by way of illustration andnot limitation, Succ-βAla, Succ-α-methyl-Ala and Succ-D-Ala.

Many cytotoxic compounds inherently have low solubility. Positivelycharged anthracyclines for example may form aggregates at highconcentration and these aggregates may induce intravenous coagulationwhen the aggregates are administered intravenously. Since manyoligopeptides have exposed, positively-charged amino termini atphysiological pH, these aggregates may form a polypositively chargedsurface in vivo and induce a coagulation cascade within a few minutes ofadministration. This has the potential for rendering any positivelycharged prodrugs that form aggregates unsuitable for therapeutic use.

One way of addressing such a potentially dangerous obstacle is toutilize the stabilizing group on the peptide chain N-terminus of anegatively charged or a neutral functionality. For example, the use ofsuccinyl as a stabilizing group on the prodrug alleviates the prodrug'sacute toxicity. This solves an important problem in the use of peptideprodrugs as practical therapies for intravenous use in humans.

Oligopeptide

Oligopeptides are generally defined as polypeptides of short length. Anoligopeptide useful in the prodrug of the invention is 3-6 amino acidsin length, however.

As indicated above, the oligopeptide has the formula X-Y, where X is aplasmin peptide substrate of 2-4 amino acids and Y is a peptidecomprising 1-2 aliphatic amino acids having large lateral chains. Thiscan also be stated as the formula or sequence (shown in the typicalamino-terminus to carboxy-terminus orientation):(AA^(x))_(m)-(AA^(y))_(n) (SEQ ID NO:1) wherein: (AA^(x))_(m) is aplasmin substrate and each AA^(x) independently represents an aminoacid; each AA^(y) independently represents an aliphatic amino acidhaving a large lateral chain; m is an integer from 2-4; and n is aninteger from 1-2.

The amino acid sequence of protease cleavage sites is conventionallydenoted as H₂N- . . . -P3-P2-P1-P1′-P2′-P3′- . . . -COOH, where the bondcleaved is between amino acids P1 and P1′ (Schlecter, et al., Biochem.Biophlys. Res. comm. 27:157-162, 1967). Plasmin is a protease withspecificity for arginine or lysine at the P1 position, with lysine beingpreferred. In several known plasmin substrates, P2 is a hydrophobicamino acid while P3 shows no apparent specificity. Accordingly,sequences containing “-amino acid P3-hydrophobic amino acid P2-lysine orarginine P1-” are expected to be good plasmin substrates (Carl, et al.,Proc. Natl. Acad. Sci. USA 77(4):2224-2228 (1980).

Therefore, X-Y in its largest embodiment, corresponds to a positionsequence —P4-P3-P2-P1-P1′-P2′-, where plasmin cleaves between the P1 andP1′ positions. The “X” plasmin peptide substrate of 2-4 amino acidscorresponds to the -P4-P3-P2-P1-section, while the “Y” 1-2 aliphaticamino acid peptide corresponds to the -P1′-P2′-section. Despite theshort length of the oligopeptide portion of the prodrug hereindescribed, the selectivity for cleavage of the prodrug by plasmin ismaintained.

Preferred Amino Acids

Unless otherwise indicated, all amino acids described herein are in theL configuration.

The following are examples of plasmin substrates suitable for use as the“X” plasmin peptide substrate of the oligopeptide. It is understood thatthis table is intended to be illustrative and not limiting in anymanner.

TABLE 1 SEQ ID NO: AA^(x4) AA^(x3) AA^(x2) AA^(x1) (if (P4) (P3) (P2)(P1) required) Leu Lys Val Leu Lys Phe Lys Ala Phe Lys Ala Lys Ala AlaLys Leu Lys Lys Glu Lys Lys Phe Glu Lys Lys SEQ ID NO:2 Glu Lys Phe GluLys Ile Glu Lys Gly Pro Lys Gly Arg Gly Gly Arg Val Gly Arg Ile Glu GlyArg SEQ ID NO:3 Pro Arg Gly Pro Arg Phe Val Arg Leu Arg Phe Arg Pro PheArg

Although many amino acids may be present in the oligopeptide portion ofthe prodrug, certain amino acids are preferred:

Suitable amino acid residues for the P4 or AA^(x4) position, include Ileand Phe residues.

Suitable amino acid residues for the P3 or AA^(x3) position, includeAla, Glu, Gly, Ile, Leu, Phe, Pro and Val residues.

Suitable amino acid residues for the P2 or AA^(x2) position, includeAla, Glu, Gly, Leu, Lys, Phe, Pro and Val residues.

Suitable amino acid residues for the P1 or AA^(x1) position, includearginine or lysine residues.

The following are examples of mono- or dipeptides useful for use as the“Y” portion of the oligopeptide. It is understood that this table isintended to be illustrative and not limiting in any manner. Unlessotherwise indicated, all amino acids are in the L configuration.

TABLE 2 AA^(y1) (P1′) AA^(y2*) (P2′)* Leu — Leu Leu *If absent, then Ycomprises one amino acid, AA^(y1).

Suitable aliphatic amino acid residues having a large lateral chain forthe P1′ or AA^(y1) and the P2′ or AA^(y2) position, include Ile, Leu,Phe and Val residues. Leu is the preferred residue.

Some preferred oligopeptides useful in the prodrug of the inventioninclude the following, shown in Table 3:

TABLE 3 AA^(x4) AA^(x3) AA^(x2) AA^(x1) AA^(y1) AA^(y2)* SEQ ID NO: (P4)(P3) (P2) (P1) (P1′) (P2′) (if required) — — Leu Lys Leu — — — Leu LysLeu Leu SEQ ID NO:4 *If absent, then Y comprises one amino acid,AA^(y1).

Linker Groups

A linker group between the oligopeptide and the therapeutic agent,although optional, may be advantageous for numerous reasons, such as thefollowing:

-   -   (1) As a spacer for steric considerations in order to facilitate        enzymatic release of the AA^(y1) or AA^(y2) amino acid;    -   (2) To provide an appropriate attachment chemistry between the        therapeutic agent and the oligopeptide;    -   (3) To improve the synthetic process of making the prodrug        conjugate (e.g., by pre-derivatizing the therapeutic agent or        oligopeptide with the linker group before conjugation to enhance        yield or specificity);    -   (4) To improve physical properties of the prodrug; and/or    -   (5) To provide an additional mechanism for intracellular release        of the drug.

Linker structures are dictated by the required functionality. Examplesof potential linker chemistries are hydrazide, ester, ether, andsulphydryl. Amino caproic acid is an example of a bifunctional linkergroup. When amino caproic acid is used in the linker group, it is notcounted as an amino acid in the numbering scheme of the oligopeptide.Accordingly, examples of suitable linker groups include amino caproicacid, a hydrazide group, an ester group, an ether group and a sulphydrylgroup.

Therapeutic Agents

Therapeutic agents that are particularly advantageous to modify to aprodrug form according to the invention are those with a narrowtherapeutic window. A therapeutic agent with a narrow therapeutic windowis one in which the dose at which toxicity is evident, by generalmedical standards, is very close to the dose at which efficacy isevident.

The therapeutic agent conjugated to the stabilizing group andoligopeptide and, optionally, the linker group to form the prodrug ofthe invention may be useful for treatment of cancer, inflammatorydisease, or some other medical condition. A series of agents can beconsidered for use in the invention. In fact, they are the same as thosethat can be used in the prodrug described in Trouet, et al., U.S. Pat.No. 5,962,216 (βAla-Leu-Ala-Leu-DOX). The main structural requirementsconcerning these agents is the presence of a reactive amino group or thepossibility to introduce such a group without critically decreasing theactivity.

In addition to therapeutics agents, the invention can also be used withagents that act as chemosensitizers and dyes. The following exemplifysuch agents and are intended to be merely illustrative and not limitingin any manner. Preferably, the agent is selected from the followingclasses of compounds: alkylating agents, anthracyclines,antiproliferative agents, camptothecins, chemotherapeutic agents,cyclosporins, dolastatins, enediynes, epipodophyllotoxins,maytansinoids, naphtalimides, platinum coordination complex, pteridines,rhodamines, sulfoximines, taxanes, taxoids, topoiosomerase inhibitors,tubulin binding agents and vinca alkaloids.

Particularly, the agent is advantageously selected from the followingcompounds, or a derivative or analog thereof: actinomycin D; alkylatingagents such as melphalan; amiodarone; anthracyclines such asdaunorubicin and doxorubicin; arabinosides such as cytosine arabinosideand adenosine arabinoside, including 1-β-D-arabinofuranosylcytosine;AT-125 or activin ((αS, 5)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid); bamipine; bleomycin; 5-bromodeoxyuridine; calicheamicin;camptothecins such as 7-amino-methylcamptothecin; L-canavanine;carboplatin; CC-1065; chlorphenoxamine; chloroquine; colchicine;combretastatin and combretastatin A₄ phosphate; coumarins such as7-amino-4-trifluoromethylcoumarin and 7-amino-4-methylcoumarin;cyclophosphamide; cyclosporins such as cyclosporin A; cytarabine;dehydrodidemnin B; dipyridamole; discodermolide; docetaxel; dolastatin10, dolastatin 11 and dolastatin 15; duocarmycin; epothilone A;etoposide and etoposide phosphate; fludarabine; 5-fluorouracil; folicacid derivatives such as aminopterin, methotrexate anddichloromethotrexate; KW-2189; 6-maytansinoids such as maytansine;mercaptopurine; methopterin; mitomycin C; naphtalimides such asamonafide; nicardipine; nitrosoureas such asN-(2-chloroethyl)-N-nitrosourea; paclitaxel; phenylenediamine mustardssuch as N,N-bis(2-chloroethyl)-p-phenylenediamine; cis-platin;podophyllotoxin and podophyllotoxin derivatives; porfiromycin;quinidine; quinine; reserpine; rhodamines such as rhodamine 123;sulfoximines such as buthionine sulfoximine, methionine sulfoximine andprothionine sulfoximine; tamoxifen; taxoids such as taxotere; topotecan;trifluoperazine; verapamil; vinca alkaloids such as vinblastine andvincristine; as well as derivatives and analogs thereof.

Some preferred prodrugs contemplated by the invention include thefollowing, shown in Table 4. Those having a “D-Ala” stabilizing groupare particularly suitable for intraperitoneal administration, whilethose having a “Succ-D-Ala” stabilizing group are particularly suitablefor intravenous administration,:

TABLE 4 Stabilizing AA^(x4) AA^(x3) AA^(x2) AA^(x1) AA^(y2) AA^(y1)* SEQID NO: Group (P4) (P3) (P2) (P1) (P1′) (P2′) Drug (if required) D-Ala —— Leu Lys Leu — DNR SEQ ID NO:5 D-Ala — — Leu Lys Leu — DOX SEQ ID NO:5D-Ala — — Leu Lys Leu Leu DNR SEQ ID NO:6 D-Ala — — Leu Lys Leu Leu DOXSEQ ID NO:6 Succ-D-Ala — — Leu Lys Leu — DNR SEQ ID NO:7 Succ-D-Ala — —Leu Lys Leu — DOX SEQ ID NO:7 Succ-D-Ala — — Leu Lys Leu Leu DNR SEQ IDNO:8 Succ-D-Ala — — Leu Lys Leu Leu DOX SEQ ID NO:8 *If absent, then Ycomprises one amino acid, AA^(y2).

Multidrug Resistance

Besides toxicity, drug resistance, a state of decreased sensitivity ofcancer cells to therapeutic agents that would ordinarily induce celldeath, is another major cause of failure in cancer chemotherapy.Resistance can be intrinsic (no response to initial chemotherapy) oracquired (selection of resistant cells in a population of cancer cellsin the course of treatment). Such acquired drug resistance is consideredthe most common reason for the failure of drug treatment in cancerpatients with initially sensitive tumors.

The most critical issue is when multidrug resistance (“MDR”) occurs. Inthis case, a given cancer cell appears resistant to a series ofdifferent anticancer agents, sometimes with quite different structuresand/or mechanisms of action. In this case, overcoming the resistancethrough combination chemotherapy is far more complicated, and researchhas focused on discovering agents that are likely to inhibit MDR.

Different mechanisms may be responsible for MDR. The most studied andperhaps the most important one involves the amplification and subsequentoverexpression of a gene (mdr1) encoding a transmembrane ATP-dependentprotein that pumps the drugs out of the cell lowering the effectiveconcentration at the target site. This pump is a 170,000 Da glycoproteincalled gp170 or P-glycoprotein. Particularly high levels of this proteinare expressed in normal kidney, liver, pancreas, small intestine, colonand adrenal gland where it might play a role in elimination or secretionmechanisms. As the P-glycoprotein transports therapeutic agents ofdifferent chemical structure (vinca alkaloids, anthracyclines,epipodophyllotoxins, actinomycin D, etc), it has been proposed to usenon-toxic compounds together with the drugs, that would act ascompetitors for capture by the pump. Several therapeutic agents havebeen tried such as verapamil, quinine or trifluoperazine, but toxicityand safety continue to remain an issue.

Accordingly, one aspect of the invention pertains to the use of theoligopeptide described herein, having a stabilizing group at itsN-terminus (or C-terminus) and verapamil, quinine, trifluoperazine oranother suitable P-glycoprotein inhibitor at its C-terminus (orN-terminus) (optionally separated by a linker group), to act as aP-glycoprotein chemosensitizer, i.e., a competitor for capture by theP-glycoprotein pump.

Another important mechanism of MDR affecting anticancer agents such asnitrogen mustard, melphalan, mitomycin, platinum derivatives, etc.involves increased intracellular reducing capacity as reflected byglutathione (“GSH”) levels. GSH normally protects cells by reacting withelectrophile derivatives and reactive oxygen species, and in cancercells, acquired resistance to alkylating agents has been associated withincreased GSH. These observations led to the development of inhibitorsof γ-glutamylcysteine synthetase (the enzyme involved in GSHbiosynthesis) as potential modulators of this type of resistance.Several interesting results have been obtained with one such compound,buthionine sulfoximine. However, due to the important physiological roleof GSH, this type of compound is not devoid of toxicity.

Whether considering the implications of P-glycoprotein-mediated orGSH-mediated MDR, it may be preferable to increase the specificity ofaction of the existing therapeutic agents, rather than to look for otherpotential modulators. The oligopeptides described herein can be used toobtain prodrugs of multidrug resistance modifiers.

It is particularly important to notice that such modulators of MDR aregenerally used not as single agents but as part of a chemotherapeuticregimen. Two types of use of the prodrugs of these MDR modulators arecontemplated by the invention: (i) use of these prodrugs together withclassical treatments, and (ii) use of these prodrugs with treatmentsbased on the less toxic prodrugs of the classically used anticanceragents described herein. The latter type of use could even lead tobetter results on resistant cells, since the prodrugs of the anticanceragents are supposed to lead to higher intracellular drug concentrations.

Accordingly, another aspect of the invention pertains to the use of theoligopeptide described herein, having a stabilizing group at itsN-terminus (or C-terminus) and buthionine sulfoximine or anothersuitable γ-glutamylcysteine synthetase inhibitor at its C-terminus (orN-terminus) (optionally separated by a linker group), to inhibit GSHproduction.

Both the (stabilizing group)-(oligopeptide)-(optional linkergroup)-(P-glycoprotein inhibitor) and the (stabilizinggroup)-(oligopeptide)-(optional linker group)-(γ-glutamylcysteinesynthetase inhibitor) described above would find utility in methods oftreating a patient.

Screening of the Prodrug

The synthesized prodrug can be tested against a test standard such asthe plasmin substrate, D-Ala-Leu-Lys, conjugated to a marker,7-amido-4-methylcoumarin. The rates of hydrolysis of the synthesizedpeptidyl prodrug and the test standard by plasmin are compared undercommon experimental conditions. The General Methods, Section N., below,provides an exemplary scheme for performing this test.

The disclosure of making and using the prodrugs taught herein provides auseful alternative to prior teachings of prodrug design. As illustratedin the examples below, the prodrugs of the invention are efficacious andwell-tolerated iii vivo in animal models. As such, the prodrugs areadvantageously utilized in therapy.

Prodrug Design

A method of designing a prodrug is another aspect of the invention andentails initially selecting a peptide of two-five amino acids, thenadding one or two amino acids having large aliphatic side chains (e.g.,Leu) to form an oligopeptide. The oligopeptide is linked at a firstattachment site of the oligopeptide to a stabilizing group that hinderscleavage of the oligopeptide by enzymes present in whole blood, anddirectly or indirectly linked to a therapeutic agent at a secondattachment site of the oligopeptide. The linkage of the oligopeptide tothe therapeutic agent and the stabilizing group may be performed in anyorder or concurrently. The resulting conjugate is tested forcleavability by plasmin under given experimental conditions. Thestandard rate of cleavage is tested on a test standard by plasmin underthe same given experimental conditions. The test standard consists of aconjugate of D-Ala-Leu-Lys and the marker, 7-amido-4-methylcoumarin. Thefirst attachment site is usually the N-terminus of the oligopeptide butmay be the C-terminus of the oligopeptide or another part of theoligopeptide. The second attachment site is usually the C-terminus ofthe oligopeptide, but may be the N-terminus of the oligopeptide oranother part of the oligopeptide. A prodrug designed by such a method isalso part of the invention.

Further, the invention includes a method for decreasing toxicity of atherapeutic agent that is intended for administration to a patient.Specifically, a modified, prodrug form of the therapeutic agent isformed by directly or indirectly linking the therapeutic agent to anoligopeptide that is cleavable by plasmin under physiologicalconditions. The prodrug provides for decreased toxicity of thetherapeutic agent when administered to the patient. The modification ofthe therapeutic agent in this manner also allows for administration ofan increased dosage of the therapeutic agent to the patient relative tothe dosage of the therapeutic agent in unconjugated form.

Pharmaceutical Compositions

The invention also includes a pharmaceutical composition comprising acompound, particularly a prodrug compound, according to the inventionand, optionally, a pharmaceutically acceptable adjuvant or vehicle.

The invention also relates to the use of the pharmaceutical compositionfor the preparation of a medicinal product intended for the treatment ofa medical condition.

The pharmaceutical composition may, for example, be administered to thepatient parenterally, especially intravenously, intramuscularly, orintraperitoneally. Pharmaceutical compositions of the invention forparenteral administration comprise sterile, aqueous or nonaqueoussolutions, suspensions, or emulsions. As a pharmaceutically acceptablesolvent or vehicle, propylene glycol, polyethylene glycol, injectableorganic esters, for example ethyl oleate, or cyclodextrins may beemployed. Isotonic saline may be part of the pharmaceutical composition.These compositions can also comprise wetting, emulsifying and/ordispersing agents.

The sterilization may be carried out in several ways, for example usinga bacteriological filter, by incorporating sterilizing agents in thecomposition or by irradiation. They may also be prepared in the form ofsterile solid compositions, which may be dissolved at the time of use insterile water or any other sterile injectable medium.

The pharmaceutical composition may also comprise adjuvants that are wellknown in the art (e.g., vitamin C, antioxidant agents, etc.) and capableof being used in combination with the compound of the invention in orderto improve and prolong the treatment of the medical condition for whichthey are administered.

Doses for administration to a patient of the compounds according to theinvention are generally at least the usual doses of the therapeuticagents known in the field, described in Chabner, et al., CancerChemotherapy (Lippincott ed., ISBN 0-397-50900-6, 1990) or they may beadjusted, within the judgment of the treating physician, to accommodatethe superior effectiveness of the prodrug formulations or the particularcircumstances of the patient being treated. Hence, the dosesadministered vary in accordance with the therapeutic agent used for thepreparation of the compound according to the invention.

Treatment of Patients with Prodrug Compound

A method for the therapeutic treatment of a medical condition thatinvolves administering, preferably parenterally and more preferablyintravenously, to the patient a therapeutically effective dose of thepharmaceutical composition is also within the scope of the invention.Thus, the method generally entails administering to the patient atherapeutically effective amount of a compound comprising:

(1) a therapeutic agent capable of entering a target cell;

(2) an oligopeptide having the formula X-Y, where X is a plasmin peptidesubstrate of 2-4 amino acids and Y is a peptide comprising 1-2 aliphaticamino acids having large lateral chains;

(3) a stabilizing group; and

(4) optionally, a linker group not cleavable by plasmin;

wherein the oligopeptide is directly linked to the stabilizing group ata first attachment site of the oligopeptide and the oligopeptide isdirectly linked to the therapeutic agent or indirectly linked throughthe linker group to the therapeutic agent at a second attachment site ofthe oligopeptide;

wherein the stabilizing group hinders cleavage of the oligopeptide byenzymes present in whole blood; and

wherein the compound is cleaved by plasmin.

The oligopeptide can also be described as having the formula orsequence: (AA^(x))_(m)-(AA^(y))_(n)(SEQ ID NO:1) wherein: (AA^(x))_(m)is a plasmin substrate and each AA^(x) independently represents an aminoacid; each AA^(y) independently represents an aliphatic amino acidhaving a large lateral chain; m is an integer from 2-4; and n is aninteger from 1-2.

The prodrug compound is useful for the treatment of many medicalconditions including cancer, neoplastic diseases, tumors, inflammatorydiseases, and infectious diseases. Examples of preferred diseases arebreast cancer, colorectal cancer, liver cancer, lung cancer, prostatecancer, ovarian cancer, brain cancer, and pancreatic cancer. Formulatedin pharmaceutically acceptable vehicles (such as isotonic saline), theprodrug compound can be administered to animals or humans in intravenousdoses ranging from 0.05 mg/kg/dose/day to 300 mg/kg/dose/day. It canalso be administered via intravenous drip or other slow infusion method.

Human patients are the usual recipients of the prodrug of the invention,although veterinary usage is also contemplated.

Diagnosis or Assay

An article of manufacture, such as a kit, for diagnosis or conducting anassay is also within the scope of the invention. Such an article ofmanufacture would preferably utilize a compound as described above,except that a marker, such as coumarin, is conjugated to theoligopeptide and stabilizing group instead of a therapeutic agent. Atleast one reagent useful in the detection of the marker is typicallyincluded as part of the kit. Thus, the article of manufacture wouldinclude the following:

(1) a compound comprising:

(a) a marker;

(b) an oligopeptide having the formula X-Y, where X is a plasmin peptidesubstrate of 2-4 amino acids and Y is a peptide comprising 1-2 aliphaticamino acids having large lateral chains;

(c) a stabilizing group; and

(d) optionally, a linker group not cleavable by plasmin;

wherein the oligopeptide is directly linked to the stabilizing group ata first attachment site of the oligopeptide and the oligopeptide isdirectly linked to the therapeutic agent or indirectly linked throughthe linker group to the therapeutic agent at a second attachment site ofthe oligopeptide;

wherein the stabilizing group hinders cleavage of the oligopeptide byenzymes present in whole blood; and

wherein the compound is cleavable by plasmin; and

(2) at least one reagent useful in the detection of the marker.

As noted above, the standard rate of cleavage is tested on a teststandard by plasmin under the same experimental conditions and the teststandard consists of a conjugate of a plasmin substrate and a marker,for example D-Ala-Leu-Lys-7-amido-4-methylcoumarin. Further, theoligopeptide can also be described as having the formula or sequence(AA^(x))_(m)-(AA^(y))_(n).

The article of manufacture may be used, for example, with patientsamples to diagnose tumors or to identify patients susceptible totreatment by prodrug therapy.

Process Chemistry General Procedures Oligopeptide: General Method forthe Synthesis of Peptides

The peptide or oligopeptide sequences in the prodrug conjugates of thisinvention may be synthesized by the solid phase peptide synthesis (usingeither Boc or Fmoc chemistry) methods or by solution phase synthesis.The general Boc and Fmoc methods are widely used and are described inthe following references: Merrifield, J. A. Chem. Soc., 88:2149, 1963;Bodanszky and Bodanszky, The Practice of Peptide Synthesis,Springer-Verlag, Berlin, 7-161, 1994; Stewart, Solid Phase PeptideSynthesis, Pierce Chemical, Rockford, 1984.

General Fmoc Solid Phase Method

Using the preferred solid phase synthesis method, either automated ormanual, a peptide of desired length and sequence is synthesized throughthe stepwise addition of amino acids to a growing chain which is linkedto a solid resin. Examples of useful Fmoc compatible resins include, butare not limited to, Wang Resins (Wang, J. Am. Chem. Soc. 95:1328, 1973and Zhang, et al., Tet. Lett. 37:5457, 1996), HMPA-PEGA resin, RinkResins (Rink, Tet. Lett. 28:3787, 1987), or a hydroxyethyl-photolinkerresin. The C-terminus of the peptide chain is covalently linked to apolymeric resin and protected α-amino acids were added in a stepwisemanner with a coupling reagent. A preferred α-amino protecting group isthe Fmoc group, which is stable to coupling conditions and can readilybe removed under mild alkaline conditions. The reaction solvents arepreferably but not limited to DMF, NMP, DCM, MeOH and EtOH. Examples ofcoupling agents are: DCC, DIC, HATU and HBTU. Cleavage of the N-terminalprotecting group is accomplished in 10-100% piperidine in DMF at 0-40°C., with ambient temperature being preferred. At the end of synthesis,the final Fmoc protecting group is removed using the above N-terminalcleavage procedure. The remaining peptide on resin is cleaved from theresin along with any acid sensitive side chain protecting groups bytreating the resin under acidic conditions. For example, an acidiccleavage condition is a mixture of TFA in DCM. If thehydroxyethyl-photolinker resin is used, the appropriate wavelength forinducing cleavage is λ365 nm ultraviolet light.

General N-cap Method via Solid Phase Synthesis

The preparation of N-terminus derivatized peptides is convenientlyaccomplished on a solid phase. When the peptide synthesis is complete,the terminal Fmoc is removed while the peptide is still on the solidsupport. The N-cap of choice is coupled next using standard peptidecoupling conditions onto the N-terminus of the peptide. On completion ofthe N-cap coupling the peptide is cleaved from the resin using theprocedure described above.

General Boc Solid Phase Method

For the solid phase method using Boc chemistry, either the Merrifieldresin or PAM resin is useful. The amino acids are coupled to the growingchain on solid phase by successive additions of coupling agent activatedBoc-protected amino acids. Examples of coupling agents are: DCC, DIC,HATU and HBTU. The reaction solvents may be DMF, DCM, MeOH and NMP.Cleavage of the Boc protecting group is accomplished in 10-100% TFA inDCM at 0-40° C., with ambient temperature being preferred. On completionof the peptide chain assembly the N-terminus protecting group (usuallyBoc) is removed as described above. The peptide is removed from theresin using liquid HF or trifluoromethane sulfonic acid in DCM. SchemesI and II illustrate the Merrifield solid phase peptide synthesis(“SPPS”) method.

N-α-Protecting Boc of the amino acid directly attached to theBoc-Leu-Merrifield Resin (1) is first cleaved in the vessel for manualSPPS by vigorous shaking with a mixture of TFA:CH₂Cl₂ (1:1, v/v) for 30min at RT to yield NH₂-Leu-Merrified Resin (2). This procedure, calledBoc-deprotection can be performed as follows (solvent volumes given for10 g of resin).

CH₂Cl₂ 60 ml  3 × 1 min TFA:CH₂Cl₂ (1:1) 50 ml  1 × 1 min TFA:CH₂Cl₂(1:1) 50 ml 1 × 30 min CH₂Cl₂ 50 ml  6 × 1 min DIPEA 5% (v/v) in CH₂Cl₂50 ml  3 × 2 min CH₂Cl₂ 50 ml  6 × 1 minStep B: N-protected Amino Acid Attachment

3 eq. of the Boc-protected amino acid to attach next, for example,Boc-Lys(Fmoc)-OH, is dissolved in CH₂Cl₂ (45 ml) and put into contactwith the resin (2) under shaking for 10 min. 3 eq. of DCC is dissolvedin CH₂Cl₂ (5 ml) and added into the reaction mixture. The latter isagitated for 2 hours at RT to produce Boc-Lys(Fmoc)-Leu-Merited Resin(3). The reaction is then checked by the ninhydrin test forcompleteness.

The ninhydrin test uses three solutions: Solution A contains 50 mgninhydrin in 10 ml EtOH; Solution B contains 80 mg phenol in 20 ml EtOH;and Solution C contains 2 ml KSCN 0.01M in H₂O in 100 ml pyridine. A fewmilligrams of the product to be tested is introduced in a test tube, andtwo drops of Solution A, two drops of Solution B, and two drops ofSolution C are added. The mixture is heated in a boiling water bath forfive min and 1 ml of EtOH is added. If the reaction is complete, theliquid remains colorless. Blue coloration of the material indicates thepresence of a free amino groups with an intensity proportional to theirconcentration.

If the ninhydrin test is negative (i.e., colorless), the Boc-attachmentstep is considered to be complete. If positive (i.e., blue), a newcoupling procedure step with the same quantity of reagents is repeated.

Step C: Attachment of Amino Acids

Attachment of subsequent amino acids involves repeating Steps A and B,as needed:

where “MR” is the Merrifield Resin.

The final coupling step with this technique generally includes aFmoc-protected amino acid, designed to terminate the sequence the besynthesized (the same proportions of reagents and procedures beingemployed as in the case of Boc-protected amino acids).

Step D: Cleavage of the Fmoc-protected Tetrapeptide(Fmoc-D-Ala-Leu-Lys(Fmoc)-Leu-OH) from the Resin

The Fmoc-protected peptide is removed from the resin by the addition ofHBr (30% in MeOH):TFA (1:1) for 30 min. This is followed by treatmentwith TFA (3×) and CH₂Cl₂ (6×).

Alternately, the Fmoc-protected peptide removal from the resin can beachieved by incubating the product in the following solutions:

CH₂Cl₂ 50 ml 1 × 10 min discard TFA:CH₂Cl₂ (1:1, v/v) 50 ml 1 × 10 mindiscard TFA 50 ml 1 × 30 min discard TFA 50 ml 1 × 30 min discardTFA:HBr (1:1, v/v) 60 ml 1 × 30 min recover TFA 50 ml  3 × 3 min recoverCH₂Cl₂ 50 ml → colorless recoverStep E: Solvent Evaporation Dissolution in MeOH and Precipitation in H₂O

The solvent is repeatedly evaporated (the solid redissolved in CH₂Cl₂)until the disappearance of an acid odor (at least 5 times). The productis dissolved in MeOH. A large quantity of ice-cold water (at least 95%of the resulting volume, w/v) is then added, the mixture stirred and theprecipitate filtered on frit glass. The Fmoc-protected peptide is thenlyophilized.

The following scheme provides the synthesis route for Compounds I andII, using DNR HCl or DNR-Leu HCl, respectively, as the startingmaterials. It is understood, however, that other anthracycline prodrugsand prodrugs of other therapeutic agents can be synthesized in a similarmanner, or by techniques that are well known to those of skill in theart.

General Procedure for the Preparation of Fmoc Oligopeptide by SolutionPhase Synthesis

Alternatively, the prodrug peptide intermediate may be made via asolution phase synthesis, utilizing either Boc or Fmoc chemistry. Thepeptide can be built up by the stepwise assembly in analogy to the solidphase method (in the N-terminal direction or in the C-terminaldirection) or through the coupling of a dipeptide with a single aminoacid.

One method of solution phase synthesis is a stepwise building up of theprodrug peptide intermediate using Fmoc chemistry. The C-terminus mustfirst be protected to reduce the formation of side products. TheC-terminal group in is typically Me, tBu, Bz or TCE. (Note when theN-cap is methyl succinyl the C-terminus group cannot be methyl.). DMF isa common solvent, as are DMSO, CH₃CN and NMP (or mixtures thereof).Pyridine, TEA or other bases may be substituted for piperidine indeprotecting the growing peptide chain protected amino terminus. HBTU isa common activating agent, but other activating agents such as DCC, DIC,DCC+HOBT, OSu, activated esters, azide, or triphenyl phosphoryl azidecan also be used. Additionally, the protected peptide acid chloride oracid bromide may be used to couple directly to the amino acid or peptidefragment. On completion of the oligopeptide assembly, the N-terminus isdeprotected and the C-terminus protected peptide is ready to accept thedesired N-cap.

General Procedure for the Preparation of N-cap Oligopeptide via SolutionPhase Synthesis

When constructing the N-capped oligopeptide by solution phase synthesis,the N-cap needs to be synthesized by a slightly modified procedure.First the C-terminus of the Fmoc oligopeptide needs to be protected withan acid labile or hydrogenation sensitive protecting group compatiblewith the selective deprotection of the C-terminus over the N-cap. Thenthe Fmoc protecting group needs to be removed from the oligopeptide toreveal the N-terminus. With the N-terminus deprotected and theC-terminus protected, the oligopeptide is reacted with the activatedhemiester of the desired N-cap. The N-cap can be activated using methodsfor activating amino acids such as DCC or HATU in base and anappropriate solvent. Alternatively, where MeOSucc is used, the couplingmay also be done via methyl hemisuccinyl chloride (or other acid halide)using an inert solvent in the presence of an organic or inorganic base,such as DIPEA, TEA or Cs₂CO₃. One example of such a synthesis can be byreacting MeOSucc and (Amino acid)_(i)-benzyl ester. The coupling methodcan be any one of the methods generally used in the art (see forexample: Bodanszky, The Practice of Peptide Synthesis, Springer Verlag,185, 1984; and Bodanszky, Principles of Peptide Synthesis, SpringerVerlag, 159, 1984. The benzyl group then can be removed by catalytichydrogenation providing the desired N-cap methylsuccinyl form of theoligopeptide. Other examples of suitable, selectively removableC-terminal protecting groups can be, but are not limited to, tBu,alkoxy-methyl and TCE. Other methods of accomplishing this step aredescribed in the literature.

The reaction conditions are well known in the art and detailed in thecitations given. The advantage of the above described methods is thefacile purification of the product produced by solution phase synthesis.

Prodrug Conjugate General Methods for the Conjugation and DeprotectionSteps

The prodrugs described herein can be synthesized by coupling an Fmocform (which means Fmoc is attached to the N-terminus of theoligopeptide) of the oligopeptide with daunorubicin, or doxorubicin, orany appropriate therapeutic agent using any of the standard activatingreagents used in peptide synthesis. The solvent may be toluene, ethylacetate, DMF, DMSO, CH₃CN, NAP, THF, DCM or any other suitable inertsolvent as is known in the art and the reagents are soluble therein.Preferred solvents include DMF and NMP. The appropriate temperaturerange is −25 to +25° C., with ambient temperature being preferred. Theactivating agent may be selected from one of the following: PyBOP, HBTU,HATU, DIC, DCC, DCC+HOBT, OSu activated esters, azide, ortriphenylphosphorylazide. HBTU or HATU are preferred activating agents.Alternatively, the acid chloride or the acid bromide of the protectedpeptide can also be used for this coupling reaction. 2-4 equivalents,advantageously 2-2.5 equivalents of a base is required for the couplingreaction. The base can be selected from inorganic bases such as CsCO₃,Na— or K₂CO₃, or organic bases, such as TEA, DIPEA, DBU, DBN, DBO,pyridine, substituted pyridines, N-methyl-morpholine etc., preferablyTEA, or DIPEA. The reaction can be carried out at temperatures between−15° C. and 50° C., advantageously between −10° C. and 10° C. Thereaction time is between 5-90 minutes and is advantageously 20-40minutes. The product is isolated by pouring the reaction mixture intowater and filtering the precipitate formed. The crude product can befurther purified by recrystallization from DCM, THF, ethyl acetate, orACN, preferably from DCM or ACN. The isolated Fmoc form of the(oligopeptide)-(therapeutic agent) conjugate is then deprotected over2-90 minutes, preferably 3-8 minutes, using a ten- to hundred-foldexcess of base at a temperature between −10° C. and 50° C. Ideally, 5-60equivalents of the base are preferred. Piperidine is the preferred baseto deprotect Fmoc groups. The deprotected amino terminus of the(oligopeptide)-(therapeutic agent) conjugate is acylated by a diacidanhydride as an activated hemiester to give the final N-cap form of theoligopeptide-therapeutic agent.

Alternatively, the final prodrug can be similarly prepared from theprotected N-cap form of the oligopeptide such as a methylhemiester formof succinyl-N-cap oligopeptide and conjugated to a therapeutic agent.

The (protected N-Cap)-(oligopeptide)-(therapeutic agent) conjugate isnow deprotected by methods compatible to the stability of thetherapeutic agent. For other therapeutic agents, benzyl protectinggroups and catalytic hydrogenation to deprotect might be chosen.

Conversion to the salt form of the negatively charged(N-cap)-(oligopeptide)-(therapeutic agent) is carried out with a solventselected from the following group: alcohol (including MeOH, EtOH orisopropanol), water, ACN, THF, diglyme or other polar solvents. Thesodium source is one molar equivalent of NaHCO₃, NaOH, Na₂CO₃, NaOAc,NaOCH₃ (in general sodium alkoxide), or NaH. An ion exchange columncharged with Na⁺ (such as strong or weak ion exchangers) is also usefulfor this last step of making the salt form of the(N-cap)-(oligopeptide)-(therapeutic agent) when appropriate. Sodium isdescribed in this application as an example only.

Generally, the prodrug may be converted to a pharmaceutically acceptablesalt form to improve solubility of the prodrug. The(N-cap)-(oligopeptide)-(therapeutic agent) is neutralized with apharmaceutically acceptable salt, e.g., NaHCO₃, Na₂CO₃, NaOHtris(hydroxymethyl)-aminomethane, KHCO₃, K₂CO₃, CaCO₃, NH₄OH, CH₃NH₂,(CH₃)₂NH, (CH₃)₃N, acetyltriethylammonium. The preferred salt form ofprodrug is sodium and the preferred neutralizing salt is NaHCO₃.

It is well documented that anthracycline type molecules, includingdoxorubicin and daunorubicin form gels in organic solvents in very lowconcentrations (Matzanke, et al., Eur. J. Biochem. 207:747-55, 1992;Chaires, et al., Biochemistry 21:3927-32, 1982; and Hayakawa, et al.,Chem. Pharm. Bull. 39:1282-6, 1991). This may be a considerable obstacleto getting high yields of clean product when making peptideanthracycline conjugates. The gel formation contributes to the formationof undesirable side reactions. One way to minimize this problem is touse very dilute solutions (1-2%) for the coupling reaction, however itis not practical in a process environment (large amounts of waste,complicated isolation). To overcome this problem, urea or otherchaotropic agents may be used to break up the strong hydrophobic andhydrogen bonding forces forming the gel. Thus if the coupling reactionis carried out in a urea-containing solvent, advantageously a 20% tosaturated solution of urea in DMF or NMP, the side reactions can be keptbelow 2% even if the concentration of reactants exceeds 10%. Thisprocedure makes the conjugation step practical at high concentrationsand produces good yields and improved purity over the procedures that donot use urea or other chaotropic agents.

General Enzyme Method

Hydrolysis of protected N-cap-oligopeptide therapeutic agents to thefull N-cap compound catalyzed by acids or bases leads to complexreaction mixtures due to the lability of many therapeutic agents evenunder moderately acidic or basic conditions. Enzymes can promote thehydrolysis without destroying the substrate or the product. Enzymessuitable for this reaction can be esterases or lipases and can be intheir natural, water soluble forms or immobilized by cross coupling, orattachment to commercially available solid support materials. Of thesoluble enzymes evaluated, Candida Antarctica “B” lipase (AltusBiologics) is especially useful example of an enzyme immobilized bycross coupling is ChiroCLEC-PC™ (Altus Biologics). Candida Antarctica“B” lipase (Altus Biologics) can be immobilized by reaction with NHSactivated Sepharose™ 4 Fast Flow (American Pharmacia Biotech). The pH ofthe reaction mixture during the hydrolysis is carefully controlled andmaintained by a pH-stat between 5.5 and 7.5, advantageously between 5.7and 6.5, via controlled addition of NaHCO₃ solution. When the reactionis completed the product is isolated by lyophilization of the filteredreaction mixture. The immobilized enzymes remain on the filter cake andcan be reused if desired.

General Allyl or Alkyl Ester Method

The prodrug can also be prepared via coupling an allyl-hemiester oralkyl-hemiester form of the N-cap oligopeptide with a therapeutic agentand then liberating the free acid from the conjugate. The coupling ofallyl-succinyl-(Amino acid)_(i)-with doxorubicin can be carried out viaany one of the oligopeptide conjugation methods.

Allyl-succinyl-(Amino acid)_(i)-doxorubicin can also be synthesized byreacting allyl hemisuccinate, which is prepared via known methods(Casimir, et al., Tet. Lett. 36:19,3409, 1995), with (Aminoacid)_(i)-doxorubicin similarly as coupling of the protected peptidylprecursors to doxorubicin was described in the previous methods.Suitable inert solvents are THF, DCM, ethyl acetate, toluene, preferablyTHF from which the acid form of the product precipitates as the reactionprogresses. The isolated acid is converted to its sodium salt asdescribed earlier. Reaction times vary between 10-180 minutes,advantageously 10-60 minutes, at temperatures between 0-60° C.,preferably 15-30° C.

Removal of the allyl or alkyl group can be done with Pd(0), or Ni(0),advantageously Pd(0) promoted transfer of the allyl or alkyl group toacceptor molecules, as it is well known in the art and documented in theprofessional literature (Genet, et al., Tet. Lett. 50:497, 1994;Bricout, et. al., Tet. Lett. 54:1073, 1998, Genet, et. al., Synlett 680,1993; Waldmann, et. al., Bioorg. Med. Chem. 7:749, 1998; Shaphiro, etal., Tet. Lett. 35:5421, 1994). The amount of catalyst can be 0.5-25 mol% to the substrate.

General Trityl or Substituted Tritel Method

The prodrug may also be synthesized by utilizing an R′-oligopeptide,where R′ is trityl or substituted trityl. The coupling ofR′-oligopeptide with a therapeutic agent can be carried out via any oneof the methods described earlier for conjugation of a protectedoligopeptide with a therapeutic agent at 30-120 minutes at 0-20° C.

Removal of trityl or substituted trityl group can be achieved underacidic conditions to give the positively charged prodrug, which isN-capped as described above. The trityl deprotection can be accomplishedwith acetic acid, formic acid and dilute hydrochloric acid.

The prodrug can be converted into (succinyl orglutaryl)-(oligopeptide)-(therapeutic agent) conjugate by reacting withsuccinic anhydride or glutaric anhydride. The solvent for coupling stepDMF, DMSO, CH₃CN, NMP, or any other suitable solvent is known in theart. Succinyl or glutaryl oligopeptide therapeutic agents can beconverted to any pharmaceutically acceptable salt.

General Inverse Direction Solid Phase Conjugation Method

The prodrug compound of the present invention can be synthesized byusing solid phase chemistry via “step wise” inverse (from the N-terminalto the C-terminal) direction methods.

One way is to use resins to immobilize a succinyl hemiester, for examplesuccinyl-mono-benzyl ester or -allyl ester. Examples of resins could beselected are Wang Resins; Rink Resins; and Trityl-, orsubstituted-trityl Resins (Chen, et. al., J. Am. Chem. Soc. 116:2661,1994; Bartos, et. al., Peptides, Proc. 22^(nd) European PeptideSymposium, 1992; and Schneider, et al., (Eds.), ESCOM, Leiden, pp. 281,1993). The immobilized ester is then deprotected and reacted with, forexample, a similarly C-terminal protected methionine. These steps arethen repeated with appropriate amino acid residues, followed by thecoupling of doxorubicin to the immobilized succinyl-tripeptide. Themolecule is then liberated from the resin by using mildly acidicconditions to form a free prodrug, such as free Succ-(Aminoacid)_(i)-Leu-DOX. Another version of phase synthesis utilizesimmobilized succinyl oligopeptide ester. This is then C-terminallydeprotected, followed by the coupling step to doxorubicin or othertherapeutic agent, and finally liberated from the resin. The acid formof the prodrug molecule may then be converted finally into its sodiumsalt as described above.

Removal of Free Therapeutic Agent

Unconjugated therapeutic agent may be present late in the process ofmaking the prodrug. For example, during the coupling step of(stabilizing group)-(oligopeptide) conjugate with doxorubicin as thetherapeutic agent, it has been found in some instances, that thereaction does not proceed completely. Initial attempts to removedoxorubicin completely can be attempted by acidic washes. In the eventthat such attempts do not result in complete removal, any remaining freetherapeutic agent can be removed by a process that utilizes scavengingresin or beads.

The crude product, which contains the intermediate and residualdoxorubicin, is dissolved in DMF and polystyrene methylisocyanate orpolystyrene sulfonyl chloride resin or beads are added. The reaction isthen stirred for 60 minutes. The free amino group of doxorubicin reactswith the isocyanate or sulfonyl chloride group on the beads to form aurea or sulfonamide derivative. The solid beads with doxorubicinattached to them are then separated from the desired product byfiltration. The desired product remains in the DMF solution. Thisapproach seems to be a very mild and effective method for removingresidual therapeutic agent from the product.

Thus, the invention includes a method of making a compound comprising:

-   -   (1) selecting an Fmoc-protected oligopeptide having a formula,        Fmoc-(AA^(x))_(m)-(AA^(y))_(n), where AA^(x), AA^(y), m and n        are as defined above;    -   (2) coupling the Fmoc-protected oligopeptide to a therapeutic        agent by activating the Fmoc-protected oligopeptide with an        activating agent in the presence of the therapeutic agent to        form an Fmoc-protected oligopeptide-therapeutic agent conjugate;    -   (3) deprotecting the Fmoc-protected oligopeptide-therapeutic        agent conjugate by contacting it with a base to form an        oligopeptide-therapeutic agent conjugate; and    -   (4) coupling the oligopeptide-therapeutic agent conjugate to a        stabilizing group to form the compound.

Alternatively, a method of making a compound comprises the followingsteps:

-   -   (1) selecting an oligopeptide having the formula,        (AA^(x))_(m)-(AA^(y))_(n), where AA^(x), AA^(y), m and n are as        defined above;    -   (2) coupling the oligopeptide to an alkyl ester-protected        stabilizing group to form an alkyl ester-protected stabilizing        group-oligopeptide conjugate;    -   (3) coupling the alkyl ester-protected-stabilizing        group-oligopeptide conjugate to a therapeutic agent by        activating the alkyl ester-protected stabilizing        group-oligopeptide conjugate with an activating agent in the        presence of a therapeutic agent to form an alkyl ester-protected        stabilizing group-oligopeptide-therapeutic agent conjugate; and    -   (4) deprotecting the alkyl ester-protected stabilizing        group-oligopeptide therapeutic agent conjugate to form the        compound.

A compound of the invention may also be made via the following steps:

-   -   (1) selecting an oligopeptide having the formula,        (AA^(x))_(m)-(AA^(y))_(n), where AA^(x), AA^(y), m and n are as        defined above;    -   (2) coupling the oligopeptide to an allyl ester-protected        stabilizing group to form an allyl ester-protected stabilizing        group-oligopeptide conjugate;    -   (3) coupling the allyl ester-protected-stabilizing        group-oligopeptide conjugate to a therapeutic agent by        activating the allyl ester-protected stabilizing        group-oligopeptide conjugate with an activating agent in the        presence of a therapeutic agent to form an allyl ester-protected        stabilizing group-oligopeptide-therapeutic agent conjugate; and    -   (4) deprotecting the allyl ester-protected stabilizing        group-oligopeptide therapeutic agent conjugate to form the        compound.

Yet another method for making a compound of the invention comprises thefollowing steps:

-   -   (1) selecting a trityl-protected oligopeptide having a formula,        trityl-(AA^(x))_(m)-(AA^(y))_(n), where AA^(x), AA^(y), m and n        are as defined above;    -   (2) coupling the trityl-protected oligopeptide to a therapeutic        agent by activating the trityl-protected oligopeptide with an        activating agent in the presence of a therapeutic agent, thereby        making a trityl-protected oligopeptide-therapeutic agent        conjugate;    -   (3) deprotecting the trityl-protected oligopeptide-therapeutic        agent conjugate under acidic conditions to form an        oligopeptide-therapeutic agent conjugate; and    -   (4) coupling the oligopeptide-therapeutic agent conjugate with        an stabilizing group to form the compound.

Another possible step in connection with any of these methods isremoving uncoupled therapeutic agent by use of scavenging resin orbeads. Further, the compound may be neutralized with a pharmaceuticallyacceptable salt if desired.

The present invention will be described in greater detail in theexamples which follow, given by way of non-limiting illustration of thepresent invention.

EXAMPLES General Methods

A. Synthesis of Amino Acid Derivatives of DNR by a ProgressiveElongation of the Peptidic Sequence

1. Products

DNR-HCl; Fmoc-protected amino acids (Novabiochem); DIPEA (98+%, Acros);DMF (Uvasol for spectroscopy, Merck); HATU (97%, Aldrich); apyrogenwater (Baxter); lactate buffer (L-Lactic acid in 85% water, Aldrich andNaOH 1N, Vel); piperidine (>99%, Fluka); chloroform (analytical reagent,Labscan); sodium sulfate (Vel); MeOH (analytical reagent, Labscan); Et₂O(>99.8%, Fluka); N-hexane (99%, Labscan); glutaric anhydride (97%,Acros); C18 ODS-A silica gel (40-60 μm, 120 Å, YMC).

2. Synthesis of (Amino Acid)_(n+1)-DNR

Piperidine (50.0 eq.) was added to a solution of Fmoc-(Aminoacid)_(n)+₁-DNR in DMF (50 ml per mmole DNR). After stirring for 5 minat RT, the mixture Et₂O:N-hexane(1:1, 6 ml per mmole DNR) was added. Theprecipitate was filtered in a glass fit, thoroughly washed with the samemixture and dissolved in chloroform (6 ml per mmole DNR). The productwas purified by silica gel column (mobile phase chloroform=MeOH, <10%),the solvent evaporated and its purity assessed by HPLC (See GeneralMethods, Section D.1.).

The residue was either used, without further purification, for furthersynthesis with Fmoc-protected amino acids or was dissolved in water byadding HCl dropwise (0.1M water solution), pH controlled (>4.0) andlyophilized.

B. TLC Method for Qualitative Analysis of DNR/DOX Peptidic Derivatives

TLC analysis was carried out on silica gel 60F-254 nm-0.25 mm plates(Merck) with DCM:MeOH:H₂O:Formic acid 88% (85:15:1:2, v/v) for elution.

C. HPLC Method #1 for Purity Analysis of Fmoc-Containing PeptidicDerivatives of DNR and DOX

The column was TSK Gel Super ODS (ref. 18197, 2 μm). Solvent A was TFA0.1% in water (w/v), and Solvent B was TFA 0.1% in ACN (v/v).

Method: 30-36% of Solvent B in 2 min, 36-41% of Solvent B in 10 min,41-90% of Solvent B in 3 min, 5 min at 90% Solvent B. Flow rate of 1.000ml/min. UV detection at 254 nm. Fluorescence detection with ex. λ of 480and em. λ of 560 nm. Purity of the compounds was assessed as thepercentage surface area of the peaks.

D. HPLC Method #2 for Purity Analysis of Peptidic Derivatives of DNR andDOX

1. DNR

The column was Luna C18 3 μm, 4.6×100 mm ID (ref. 00d-4251-e0). SolventA was TFA 0.1% in water (w/v), and Solvent B was TFA 0.1% in ACN (v/v).

Method: 31% of Solvent B for 5 min, 31-43% of Solvent B in 3 min, 43-90%of Solvent B in 0.5 min, 2.5 min at 90% Solvent B. Flow rate of 1.500ml/min. UV detection at 254 nm. Fluorescence detection with ex. λ of 480and em. λ of 560 nm. Purity of the compounds was assessed as thepercentage surface area of the peaks.

2. DOX

The column was Symmetry shield waters RP8 3.5 μm, 4.6×150 mm ID (PartAWT094269). Solvent A was 80% formate ammonium in water (w/v) 20 mMpH4.5+20% acetonitrile (v/v), and Solvent B was 20% formate ammonium inwater (w/v) 20 mM pH4.5+80% ACN (v/v).

Method: Column is equilibrated with solvent A for 7 min. Gradient of100% Solvent A to 100% solvent B in 30 min. Flow rate of 1.000 ml/min.UV detection at 254 nm. Fluorescence detection with ex. λ of 480 and em.λ of 560 nm. Purity of the compounds was assessed as the percentagesurface area of the peaks.

E. Cultured Cells

MCF-7/6 cultured cells were graciously provided by Professor M. Mareelfrom the Ghent University (Belgium).

The B16-B16 cell line was obtained from the ATCC.

LS-174-T, the trypsinized variant of LS-180 (CL-187) colonadenocarcinoma line, was obtained from ATCC (CL 188)

F. Culture Medium and Routine Cell Culture

DMEM-F12 (Gibco) culture medium was used for all types of culturedcells. It was completed with 10% BFS (Gibco). A so-called “defined”culture medium was used for the preparation of the cell-conditionedmedium.

1. Serum-containing Medium

Serum-containing medium was generally composed of a basic culture mediumcompleted with 10% inactivated BFS. Inactivation was accomplished byincubating the serum at 56° C. for 30 min in a water bath. L-Glutamine(2 mM) was also added. Some culture media already contained a stableform of the L-glutamine equivalent, called glutamax. 100 IU/ml ofpenicillin and 0.1 mg/ml of streptomycin could also be added to thecultured cells to avoid bacterial contamination.

2. Defined Medium

The medium was prepared with DMEM-F12 without phenol red, and completedwith the following products: 10 μg/ml human apotransferrin, 200 μg/mlBSA, 1 μg/ml insulin and 10 nM of 17-β-estradiol.

No serum was added to the medium.

3. Routine Cell Culture

All cell types were commonly cultured in sterile TC/PS flasks,maintained in a standard incubator at 37° C. (95% O₂ and 5% CO₂). Theculture medium was changed every 2-3 days.

Confluent cells were harvested for transfer into new flasks. This wasdone by rinsing the confluent cell layer with PBS containing 0.53 mMEDTA, completed with 0.25% trypsin. When harvested (from 5-10 min ofincubation with the trypsin-containing solution), the cells weresuspended in a fresh serum-containing culture medium and centrifuged at300 g for 10 mm. The pellet was resuspended in fresh medium anddistributed into new flasks.

G. Preparation of Medium Conditioned by MCF-7/6 Cancer Cells (“CM”)

MCF 7/6 cells were grown until subconfluence (not entirely continuouscell layer) in complete medium in 175 cm² TC/PS flasks. The cells werethen washed with PBS (3×), 15 ml of defined medium was added to eachflask, and the cells incubated for 24 hr at standard conditions.

The preparation of the CM, concentrated 20× was as follows. The mediumwas recovered, cooled down to 4° C. and centrifuged at 300×g for 10 minto remove suspended cells. The medium was then concentrated 20× using anAmicon concentrator (Millipore) containing membranes with a cut off at10 kDa (YM10-Millipore).

H. Preparation of Drug Solutions

Compounds to be tested in biological experiments (stability, uptake,cytotoxicity tests or in vitro injections) were dissolved in water (1/10 of the expected volume) and further diluted with 0.9% (w/v) NaCl.The concentration of these solutions was checked (A_(475nm)determination for anthracyclines) after sterilization by filtration(0.22 μm) and adjusted to the final required value using sterile 0.9%(w/v) NaCl.

I. Test of Stability of Oligopeptidic Derivatives of Anthracyclines inBlood and Conditioned Medium

Compounds were mixed with fresh blood of healthy volunteers collected inheparinized tubes or CM (4° C., ice water bath) to obtain the desiredfinal dilution. Compounds were extracted immediately after being mixedin (time 0) as well as after desired intervals of incubation (37° C.,ice water bath).

1. Incubation (Example)

810 μl of blood+90 μl of the compound tested at 172.4 μM or 540 μl of CM(concentrated 20×)+60 μl of the compound at 172.4 μM were mixed inEppendorf tubes. CM (20×) was made 10% (v/v) of phosphate buffer (1M pH7.4) before use.

2. Extraction (Example)

In glass tubes of 10 ml was mixed 1.8 ml of chloroform:MeOH (4:1, v/v).Compounds in which the amino group was replaced by a free carboxy group(succinylated prodrugs for example) were extracted with 600 μl of citricbuffer (citric acid 0.5M water solution, pH adjusted to 3.5 with NaOH).Compounds were extracted immediately as well as after desired periods ofincubation (3×25 μl). The sample (500 μl) and 100 μl of the internalstandard at 3.5 μM were added to the mixture (commonly DOX-HCl for DNRderivatives and L-prolyl-DNR for DOX derivatives). 600 μl of boratebuffer (0.5M pH 9.8) was added and immediately vortexed. The tubes werecentrifuged at 1500 g for 5 min. The organic phase (1 ml) was recoveredin glass tubes and dried under nitrogen flow. A mixture of TFA 0.1% inH₂O (70%, v/v) and TFA 0.1% in ACN (30%, v/v) was added to each tube(500 μl).

The compounds were dissolved by ultrasonication for 15 sec between (100W). The solution was recovered and filtrated (0.22 μm filters) for HPLCanalysis (See General Methods, Section J.).

J. HPLC Method #3 for Analysis of DNR and its Peptidic DerivativesDuring Experiments In Vitro

The column was TSK Gel Super ODS (ref. 18197, 2 μm). Solvent A was TFA0.1% in water (w/v), and Solvent B was TFA 0.1% in ACN (v/v).

Method: 30% of Solvent B (v/v) for 6.5 min, 30-90% of Solvent B (v/v) in1 min, 2 min at 90% Solvent B. Flow rate of 1.500 ml/min. UV detectionat 254 nm. Fluorescence detection with ex. λ of 480 and em. λ of 560 nm.Purity of the compounds was assessed as the percentage surface area ofthe peaks.

K. Synthesis of Fmoc-protected Oligopeptides by the Merrifield SPPSMethod

The Merrifield SPPS method is described in The Chemistry ofPolypeptides, P. G. Katsoyannis Ed., Plenum Press, New-York, pp.336-361, 1973. This method includes three major steps and is shown inSchemes I and II. The completeness of the reaction was measured by theninhydrin test, as described above in Scheme I.

L. HPLC Method #4 for Analysis of Fmoc-containing Oligopeptides

The column was TSK Gel Super ODS (ref. 18197, 2 μm). Solvent A was TFA0.1% in water (w/v), and Solvent B was TFA 0.1% in ACN (v/v).

Method: 0-70% of Solvent B in 30 min. Flow rate of 1.200 ml/min. UVdetection at 254 nm. Fluorescence detection with ex. λ of 480 and em. λof 560 nm. Purity of the compounds was assessed as the percentagesurface area of the peaks.

Sample preparation was as follows. Peptide samples for HPLC wereprepared by dissolving the products in acetic acid (96%, v/v) or MeOH(+t° C.) at approximately 10 mg/ml and filtered on a 0.22 μm filter. Notmore than 10 μl of such sample was then injected for analysis.

M. Synthesis of Peptidic Compounds by Coupling of Fmoc-protectedOligopeptides to Anthracyclines

1. Products

DOX-HCl; DNR-HCl; L-Leu-DOX-HCl; L-Leu-DNR-HCl;Fmoc-D-Ala-Leu-Lys(Fmoc)-Leu-OH SPPS (See General Methods, Section K);HATU; DIPEA; DMF; apyrogen water; lactate buffer; piperidine; CDM(analytical reagent, Labscan); chloroform; sodium sulfate; MeOH; Et₂O;N-hexane; EtOH (for synthesis, Merck); formic acid (GR, Merck); ACN(analytical reagent, Labscan); TFA (free acid, Sigma); C18 ODS-A silicagel (40-60 μm, 120 Å, YMC).

2. Synthesis of (Fmoc)_(n)-peptide-anthracycline

Anthracycline-HCl (1.0 eq.) and the Fmoc-protected peptide (1.2 eq.)were dissolved in DMF (50 ml per mmole of anthracycline). After additionof DIPEA (2.0 eq.), the mixture is stirred for 15 min at RT (in thedark). A solution of HATU (1.1 eq.) in DMF (20 ml per mmole ofanthracycline) was added. After the mixture was stirred for 2 hours,cold water (4° C., 135 ml per mmole of anthracycline) was added andstirred an additional 30 min. The precipitate was filtered onqualitative paper (Whatman 1), washed with water (20 ml per mmole DNR),2% lactate buffer (pH 4, 2×30 ml per mmole DNR), then water (2×30 ml permmole DNR). The solid was dissolved in water and lyophilized to giveFmoc-peptide-anthracycline. Purity was assessed by TLC (See GeneralMethods, Section B.) and HPLC (See General Methods, Section C.).

3. Synthesis of Peptide-anthracycline Lactate

Piperidine (50.0 eq. per Fmoc-group) was added to a solution ofFmoc_(n)-anthracycline in DMF (50 ml per mmole DNR). After stirring for5 min at RT, the reaction mixture was cooled (ice salt bath) andpre-cooled (4° C.), 10% lactate buffer pH 3.0 was added. The aqueoussolution was extracted with DCM (3×100 ml per mmole anthracycline) andpurified by solid phase extraction (YMC gel, 25 g per mmole ofanthracycline). The MeOH was removed and the residue was dissolved inwater and lyophilized to give peptide-anthracycline lactate. Its puritywas assessed by HPLC (See General Methods, Section D.1. or D.2.).

N. Plasmin Assay

This assay is based on the proteolytical cleavage ofD-Ala-Leu-Lys-7-amido-4-methylcoumarin by plasmin with restitution of afluorogenic compound.

Materials: D-Ala-Leu-Lys-7-amido-4-methylcoumarin (Sigma), 2 mM stocksolution in DMF, as substrate; TES buffer; control enzyme solution(Sigma) or sample; PBS; and 7-amido-4-methylcoumarin (Sigma).

In an eppendorf tube, 50 μl of the substrate at a concentration of 50 μMin DMF were mixed with 900 μl of TES buffer and 50 μl of anenzyme-containing solution. The mixture was incubated for 1 hour at 25°C. Fluorescence was determined (λ_(exc)=350 nm, λ_(em)=496 nm;fluorometer standardized with a 10 nM solution of7-amido-4-methylcoumarin in PBS and the whole mixture excluding theenzyme). A dosage curve was done in parallel with known plasminconcentrations (at least five ½ dilutions). Plasmin activity wasdetermined and expressed in IU/ml; μmole substrate transformed/min×ml.

O. Chemotherapy Studies (General Procedure)

Animals were kept under pathogen-free conditions with food and watersupplied ad libitum, and kept for about one week before implantation ofthe tumors. Fragments (˜2 mm) of tumors grown in nude mice wereimplanted subcutaneously in both flanks of the mice. Tumors weregenerally allowed to grow to attain a diameter of 5-6 mm each(approximately two weeks).

The mice weighed 20.0-25.0 g at the beginning of the study. They wereselected and assigned to groups in order to have equally distributedtumor volumes in the different groups. In each group, mice were labeledindividually. Treatments were assigned randomly to those groups.

All dosing solutions were prepared the day of injection by dissolutionin water ( 1/10 of the expected final volume) and further dilution with0.9% (w/v) NaCl. The concentration of these solutions was checked(A_(475nm) determination) after sterilization by filtration (0.22 μm)and adjusted to the final required value using sterile 0.9% (w/v) NaCl).

Treatments were administered by the i.p. route. The mice received aconstant volume (10 μl/g) of either saline (control group) or drug.Mortality and clinical signs were recorded up to 4 hours post-treatmenton dosing days, and daily thereafter. Their behavior was equally noted.Individual body weights were generally recorded daily until day 14 andat least twice a week thereafter.

Tumor growth was monitored by two-dimensional measurements usingcalipers and a precision of 0.5 mm. Tumor volumes were calculatedaccording to the following formula:V _(t)=[length×(width)²]/2

Median relative tumor volumes (RTV) were calculated as tumor volumesdetermined at individual days divided by tumor volumes on day 0. Fortreated groups, growth inhibition was estimated as the percentage ratioof median RTV of treated (T) mice versus controls (C) on each day oftumor treatment.T/C(%)=[Median RTV (treated group)/Median RTV (control group)]×100

The minimal T/C value (%) for each treatment was used as a parameter formaximum efficacy. Duration of growth inhibition was assessed byconsidering growth delays (T−C) for one (200%) and two (400%) doublingsof the median RTV).

To determine the statistical significance of any difference in medianRTV between two groups, the Mann-Whitney test was used. The test wasconducted at a p level of 0.05 (two tailed) using the GraphPad Prism3.00 software.

P. Lethality Studies (General Procedure)

The percentage of surviving mice and the mean body weight of each dosegroup was plotted as a function of time. Cumulative mortality on day 14and day 28 was also plotted as a function of the dose level for thepurpose of IC₅₀ determination. IC₅₀ values were determined fromsigmoidal regressions performed with the GraphPad Prism 3.00 software.

EXAMPLE 1 Synthesis of DNR and DOX Prodrugs

D-Ala-Leu-Lys-Leu-DNR (Compound I, DNR; SEQ ID NO:5) andD-Ala-Leu-Lys-Leu-Leu-DNR (Compound II, DNR; SEQ ID NO:6) weresynthesized by covalent coupling of the protected peptide on theanthracyclines or on their L-Leucyl derivatives.

Compound I was synthesized by linking the peptide D-Ala-Leu-Lys-Leu toDNR. The Fmoc-protected tetrapeptide Fmoc-D-Ala-Leu-Lys(Fmoc)-Leu-OH wasproduced by SPPS using Boc-Leu-Merrifield resin, N-α-Boc-N-ε-Fmoc-Lys-OH(“Boc-Lys(Fmoc)-OH”), N-α-Boc-Leu-OH (“Boc-Leu-OH”) andN-α-Fmoc-D-Ala-OH (“Fmoc-D-Ala-OH”), with DCC as the coupling agent (SeeScheme I).

The Fmoc-protected peptide was then coupled to the amino-group of DOX orDNR or of their L-Leucyl derivative using HATU as the coupling agent andpiperidine for Fmoc deprotection (See Scheme II).

Progress of the coupling and deprotection reactions was followed by TLC(CHCl₃:CH₃OH:H₂O; 120:20:1 by volume) and HPLC (See General Methods,Section C.). The final products were analyzed by mass spectrometry andHPLC (See General Methods, Section D.), electrospray and NMR. Details ofthe general procedure of the peptide synthesis are given above inreference to Schemes I and II. The HPLC method adapted for analysis ofFmoc-containing oligopeptides is described in General Methods, SectionL.

Following these methods, similar compounds were synthesized using theanthracycline, DOX: D-Ala-Leu-Lys-Leu-DOX (Compound I, DOX) andD-Ala-Leu-Lys-Leu-Leu-DOX (Compound II, DOX).

The use of Fmoc for the protection of the amino groups of D-alanine andL-lysine assured an excellent stability of the growing peptide duringBoc-deprotection and final cleavage of the peptide from the resin. Atthe same time, both of these protecting groups were easily removed witha one-step deprotection by piperidine (100 eq.). This simple approachallowed for synthesis of all designed compounds in only two steps withfairly good yields. The use of a unique batch of tetrapeptideD-Ala-Leu-Lys-Leu-OH for synthesis of four compounds assured theproduction of a homogeneous material for subsequent in vitro and in vivoexperiments. Final purity of the synthesized compounds ranged from90-97% according to HPLC analysis and was considered acceptable for invitro and in vivo tests. Analytical results confirmed the expectedmolecular masses and structures.

EXAMPLE 2 Plasmin Activity in Human Blood and Blood Stability ofCompounds I, DNR and II, DNR

Determination of plasmin activity was done as described in GeneralMethods, Section N. A solution of D-Ala-Leu-Lys-7-amido-4-methylcoumarinwas mixed with human blood to the final concentration of 5 μM andincubated at 37° C. for 15 min and the supernatant taken for analysis.Blood in the control solution was replaced by water. Solutions ofCompounds I, DNR and II, DNR in water (17.24 mM) were added to thefreshly collected (on citrate) whole human blood from healthy donors(final concentration; 0.06 mM for Compound I, DNR; 0.18 mM for CompoundII, DNR) and incubated at 37° C. The compounds were extracted and HPLCsamples prepared as described in General Methods, Section I. DOX wasused as the internal standard. The samples were analyzed by the HPLCmethod described in General Methods, Section J.

No traces of plasmin-like activity were discovered in human blood withthe synthetic substrate, D-Ala-Leu-Lys-7-amido-4-methylcoumarin. Valuesobtained after 1 hour with incubation of substrate dilution with bloodat 37° C. did not differ from those obtained for the negative controlsolution.

Compound I, DNR (0.06 mM) remained perfectly stable in vitro, afterincubation with human blood for 1 hour. Small peaks of productsresulting from a non-specific degradation of the compound (called“aglycones” and not quantified) were observed after 8 hours. Noanthracycline derivatives were liberated from the compound during thisincubation time.

Compound II, DNR (0.18 mM) exhibited excellent stability in vitro, inthe presence of human blood, with ±5.0% of L-Leu-DNR liberated after 24hours.

Both compounds showed an excellent stability in human blood during theincubation at 37° C. A small quantity of degradation products after 1hour of incubation were not due to the action of plasmin. After 24 hoursof incubation of Compound II, DNR, only 5% of the incubation mixture wascomposed of L-Leu-DNR These results correspond to those obtained withD-Ala-Leu-Lys-7-amido-4-methylcoumarin. This synthetic substrate ofplasmin remained entirely stable during 1 hour of incubation at 37° C.

EXAMPLE 3 Hydrolysis of Compounds I, DNR and II, DNR by Human Plasmin

Compounds I, DNR and II, DNR (0.17 mM), as well asD-Ala-Leu-Lys-7-amido-4-methylcoumarin (0.17 mM) were incubated at 25°C. in the presence of 0.1 units of human plasmin in a 50 mM, pH 8.0 TESbuffer. For Compounds I, DNR and II, DNR, HPLC samples were prepared andanalyzed by HPLC (See General Methods, Section J). In case ofD-Ala-Leu-Lys-7-amido-4-methylcoumarin, after 1 hour of incubation, thefluorescence of the resulting solution was determined at λ_(exc)=350 nm,λ_(em)=496 nm (See General Methods, Section N.).

Compound II, DNR (0.05 mM) was incubated at 25° C. in the presence of0.01, 0.05 or 0.1 units of human plasmin in a 50 mM, pH 8.0 TES buffer.After 15, 45 and 60 min of incubation, triplicate aliquots were removedand the drugs and metabolites extracted prior to HPLC analysis (SeeGeneral Methods, Section J.).

Compounds I, DNR and II, DNR (17.24 μM) were incubated in a mediumfreshly conditioned by the cultured B16-B16 cells (20 timesconcentrated) for 2 hours at 37° C. After that time, triplicate aliquotswere removed and the drugs and metabolites extracted prior to HPLCanalysis (See General Methods, Section J.).

The results indicated that Compound I, DNR was relatively weaklyhydrolyzed in vitro by human plasmin (approximately 2.75 nmol/min/unit).At the same time, the in vitro hydrolysis of Compound II, DNR was atleast 10 times more rapid (27.90 nmol/min/unit). Enzymatic hydrolysis ofD-Ala-Leu-Lys-7-amido-4-methylcoumarin monitored by7-amido-4-methylcoumarin liberation under the same conditions gave avalue of 20.40 nmol/min/unit.

Hydrolysis rate was found to be directly proportional to time and enzymeconcentration with all plasmin dilutions. No hydrolysis of any of thecompounds in the medium conditioned by B16-B16 cells was observed. Onlysmall quantities of products resulting from a non-specific degradationwere observed.

The results confirmed the role of an amino acid spacer in the efficienthydrolysis of amino acid derivatives of anthracyclines by plasmin. Thekinetics of hydrolysis were shown to be linear andconcentration-dependent. Negative results with the medium conditioned byB16-B16 cells obtained with 7-amido-4-methylcoumarin and Compounds I,DNR and II, DNR, indicate the absence of important plasmin-like activityin it, which can possibly be attributed to a cell surface-associatedplasmin formation or to the absence of specific conditions forplasminogen activation in vitro.

EXAMPLE 4 In Vitro Accumulation by Cultured Tumor Cells

Compound I, DNR was incubated in vitro at 37° C. in the presence ofMCF-7/6 human mammary carcinoma cells and the intracellular accumulationof the compound or its potential metabolites were analyzed by HPLC andfluorimetry, after extraction of the drugs from the cells.

Compound I, DNR was dissolved in distilled water at 17.24 μmole/ml andthen diluted to obtain 0.02 mM in DMEM-F12 culture medium supplementedwith 10% fetal calf serum (glutamax-I). Four ml of medium was poured in25 cm² flasks (ref.3014. FALCON), and the cells were incubated for 30minutes, 1, 3, 6 and 24 hours at 37° C. Then the cells were washed threetimes with PBS, and the cells were scraped from the flasks, andsonicated for 30 seconds at 100 Watts in a ice bath. Aliquots of 500 μlfrom each sample were then extracted with 1.8 ml of achloroform:methanol mixture (4:1 by volume) to which 0.1 ml of 0.2 Mborate buffer at pH 9.2, containing DOX at 3.4 mmole/ml as internalstandard, were added. After stirring, the organic layer was dried andresuspended in 400 μl of distilled water, sonicated 5 minutes at 100Watts in a ice-bath and filtered through a 0.22 μm filter(Waters-Millipore, MILLEX-GV) and then analyzed by HPLC.

On a aliquot of 100 μl, the protein content of the cell homogenate wasdetermined. The results indicated that the accumulation of Compound I,DNR by cultured MCF-7/6 mammary carcinoma cells after 24 hours wasextremely weak, representing approximately 0.1 μg/mg of cell proteins.

EXAMPLE 5 Uptake of N-Leu-Leu-DNR and Compounds I, DNR and II, DNR byB16-B16 Cells

This experiment was conducted to evaluate whether the prodrugs were ableto enter cultured cells in their non-hydrolyzed forms. In addition, itwas desired to estimate the capacity of the prodrugs to inducedetectable levels of products of their enzymatic hydrolysis (DNR and itsamino acid derivatives) inside B16-B16 cells. N-Leu-Leu-DNR was chosenas a reference drug since it is the product that is directly liberatedby plasmin from Compound II, DNR.

Confluent B16-B16 melanoma cell cultures were incubated in the presenceof 17.24 μM dilutions in the serum-containing DMEM-F12 medium of eitherCompound I, DNR, Compound II, DNR, or N-Leu-Leu-DNR. At determined timepoints (1, 3, 5 and 24 hours), the cells were washed, harvested andlysed by ultrasonication. The concentration of the drugs and metabolitesin the cell lysates were determined after extraction and HPLC analysis(See General Methods, Section J.). The amounts of anthracyclinederivatives found per mg of protein were then compared.

The intracellular level of N-Leu-Leu-DNR after 3 hours of incubationwith 17.73 μM of N-Leu-Leu-DNR was 7.74±0.75 nmol/mg cell protein, thatof DNR was 0.67±0.12 nmol/mg cell protein, and that of L-Leu-DNR was81.22±6.66 nmol/mg cell protein. After 24-hours, DNR attained 1.60±0.77nmol/mg cell protein, L-Leu-Leu-DNR attained 1.04±0.10 nmol/mg cellprotein, and L-Leu-DNR attained 98.57±16.76 nmol/mg cell protein.

In the case of Compound I, DNR, three major derivatives were presentintracellularly at every point of incubation. A low level ofnon-hydrolyzed prodrug was detected. It grew up to three hours ofincubation and remained stable afterwards. Its level was situated at0.26±0.02 nmol/mg cell protein after 24 hours. On the other hand, thequantity of L-Leu-DNR continued to rise over a 24 hour period, to anintracellular level of 0.53±0.03 nmol/mg cell protein. DNR began toappear from the first hour (0.015±0.002 nmol/mg cell protein) to attainthe level of 0.023±0.002 nmol/mg cell protein.

The level of Compound II, DNR detected did not considerably change intime and had the approximate value of 0.3 nmol/mg cell protein (startingfrom the first hour of incubation). A major metabolite found wasL-Leu-DNR with quantities rising constantly during the study. The finalquantity of this compound was 12.30±0.67 nmol/mg cell protein. Smallquantities of DNR were also detected. Their level was 0.34±0.01 nmol/mgcell protein after 5 hours and 0.56±0.06 nmol/mg cell protein at the endof the incubation.

The absence of uptake of the non-hydrolyzed Compounds I, DNR and II, DNRis extremely important, along with their blood stability and sensitivityto plasmin action. The combination of these three properties allowsthese prodrugs to be classified as potential extracellularly tumoractivated prodrugs.

Comparison of the kinetics of appearance of L-Leu-DNR and DNR duringincubation of the tetra- and pentapeptidic derivatives with B16-B16cells showed L-Leu-DNR being the major product and DNR displaying quitesmall and very slowly increasing quantities. At the same time, the levelof L-Leu-DNR by Compound II, DNR was much higher as compared withCompound I, DNR (45.1-fold higher after 5 and 23.4-fold after 24 hoursof incubation). No traces of N-Leu-Leu-DNR were found inside cellsincubated in the presence of the pentapeptidic compound.

The absence of hydrolysis of Compound II, DNR in the medium conditionedby B16-B16 could be explained by the localization of plasmin-likeactivities on the cellular surface.

The pentapeptidic prodrug was shown to be more efficient that itstetrapeptidic counterpart in inducing intracellular L-Leu-DNR. This dataconfirmed the previously observed better enzymatic activation byCompound II, DNR in vitro.

EXAMPLE 6 Acute Toxicity of Compound II, DOX in OF-1 Mice

Compound II, DOX was compared with DOX for determining its acutetoxicity in OF-1 mice after intraperitoneal (i.p.) injection.

Male OF-1 mice (5 weeks old upon delivery), were kept for one week underlaboratory conditions before the experiment. The body weight of theanimals was approximately 30-35 g at the beginning of the test. Theanimals were divided in six groups (5 mice per group) for toxicitystudies and marked individually. Each group received five i.p.injections of respectively 0.9% (w/v) NaCl (control group), or CompoundII, DOX at 17.24, 25.86, 34.48, 43.1 and 60.34 μmol/kg on days 0-4.

Mortality and clinical signs were recorded up to 4 hours post-treatmenton dosing days, and daily thereafter. Individual body weights wererecorded daily until day 14 and at least twice a week thereafter. Thepercentage of surviving mice and the mean body weight of each dose groupwere plotted as a function of time. Cumulative mortality on day 14 andday 28 was also plotted as a function of the dose level for the purposeof LD₅₀ determination. LD₅₀ values were determined by regressionanalysis.

All animals survived to day 7. No lethality at all was observed in thecontrol group of animals to the end. In terms of body weight, thecontrol animals grew to reach a mean of 119.1% on day 35 (compared withday 0). All animals remaining from groups having received Compound II,DOX at 25.86 μmol/kg.

Important body weight mean values changes were observed starting fromthe dose of 34.48 μmol/kg. All of these groups attained the criticalthreshold of 20% of loss as compared to the initial body weight. In thecase of groups having received 43.1 and 60.34 μmol/kg, this arrivedalready after the first week of and for those with 34.48 μmol/kg, afterthe second one.

Clinical signs of toxicity observed for Compound II, DOX were the sameas those caused by DOX: piloerection, tremor, motor disturbances, stuporand finally, paralysis of the hind legs. Appearance of all clinicalsigns correlated with the importance of the dose administered and servedas negative prognostic factors to the survival of the animals.

The LD₅₀ value calculated on day 28 was 34.5 μmol/kg. The LD₅₀ value forDOX obtained under the same conditions was known to be 4.1 μmol/kg.

To be considered as a prodrug, a new compound should possess a decreasedgeneral toxicity. Thus, the expected LD₅₀ in a toxicity study should behigher for a conjugated drug as compared with the parent drug. CompoundII, DOX has shown an LD₅₀ value 8.4-fold higher than that of DOX underthe same conditions (34.5 as compared to 4.1 μmol/kg). The toxicitysigns observed during this study with Compound II, DOX arecharacteristic of DOX, severe weight losses and neurotoxicity.

EXAMPLE 7 Chemotherapeutic Activity of Compound II, DOX in the B16-B16Murine Melanoma Model

The mouse B16-B16 melanoma model was chosen for the first chemotherapyexperiment with Compound II, DOX.

Male C57-B16 black mice were 5 weeks old upon delivery. The body weightof the animals was approximately 20 g at the beginning of the test (SeeGeneral Methods, Section O).

The animals were divided in eight groups (5 mice per group). Confluentmurine melanoma B16-B16 cells (See General Methods, Section E.) werewashed twice with PBS and EDTA (See General Methods, Section F.) twiceand detached with trypsin (2 min incubation at 37° C.), that wasthereafter neutralized by DMEM-F12 (containing 10% of calf serum). Thecells were centrifuged for 5 min at 300 g. The pellet was resuspendedtwice in the DMEM-F12 without serum and centrifuged 5 min at 300 g.

The number of cells per ml of DMEM-F12 was then estimated (Bruckerchamber) and the dilution of 2.5×10⁵ cells/ml prepared. Each animalreceived 200 μl of this suspension (5.0×10⁴ cells) in its tail vein. Theday of injection was considered day 0 of the study. Each group receivedtwo i.p. injections of respectively 0.9% (w/v) NaCl (control group), DOXat 5.2 μmol/kg and at 8.6 μmol/kg, or Compound II, DOX at 34.5 μmol/kgand at 69.0 μmol/kg on days 1 and 3. Animals were weighed on days 0, 1,3, 6, 8 and 14. Their behavior and eventual clinical signs were alsonoted. All animals were sacrificed on day 14 and autopsied.

The lung lobes were fixed in a Bouin's solution (picric acid:formaldehyde, 40%: acetic acid, 15:5:1, v/v). Every lobe immersed in thefixing solution in a Petri flask was uniformly lighted against an opaqueblack background. Pictures of the front and back side of every lobe weretaken with the help of a light microscope, scanned and digitized. Therelative surface occupied by B16-B16 colonies was then estimated withthe Scion Image Program.

The individual mean values were analyzed with the Scheffe's F-test toevaluate the statistical significance of differences observed betweenthe groups. Each drug-treated group was compared to the control groupand DOX-treated groups were compared to Compound II, DOX-treated groups.

No important weight changes were observed during the first week of theexperiment for either group. The control group had better values up today 8 (with a maximum at day 6 situated at 106.3% of day 0). Thatdropped to 101.6% at the end of the study.

In the case of the group treated with DOX at 5.2 μmol/kg, this parameterdid not change substantially during the experiment, with a slightincrease at day 14 (104.7%). Such an increase was more important for thegroup treated with DOX at 8.6 μmol/kg with 110% of initial mean weightat the end. The group having received Compound II, DOX at 34.5 μmol/kgfollowed a positive evolution with 104.5% on day 14. The group treatedwith Compound II, DOX at 69.0 μmol/kg remained more or less stable, witha slight tendency to lose weight up to day 8 (92.3%) broken afterwardswith an increase up to 98.7% at the end of the experiment.

Non-treated animals from the control group had, with no exception, largemerging black spots on every lobe analyzed. The ratio of the surfaceoccupied by B16-B16 colonies to the non-affected one varied largely fromone animal to another with a mean value of 45.7±12.6%. It was similarfor the group treated with 5.2 μmol/kg of DOX (44.0±6.3% of surfaceoccupied by metastases). The value obtained for the group havingreceived DOX at 8.6 μmol/kg was 23.4±5.7%.

Compound II, DOX at 34.5 μmol/kg had a significant influence on thespread of lung metastases with a decrease to 8.2±1.8% (P<0.01). The sameprodrug at 69.0 μmol/kg provided 1.5%±0.6% of surface affected(P<0.001). It was noteworthy that there were still a large number ofblack spots scattered all over the lobe surface in this group. Thedifference was in the small size. The large size of metastases in thecontrol group and the two groups treated with DOX and their subsequentmerging did not permit any precise quantification of their number. Inthe case of animals treated with prodrugs, their number did not show anysignificant difference between the groups with values of approximately800 per animal.

The study was designed to compare the chemotherapeutic efficacy ofCompound II, DOX to that of DOX using a model involving a metastaticspread of cancer cells into the lungs. The B16-B16 murine melanoma modelhas the advantages of presenting the opportunity to quantify themetastatic progression, and the cells present a well-describedimplication of the plasminogen-plasmin system during their growth andmetastasis.

In toxicity studies with high doses, weight loss is a direct consequenceof drug toxicity. Chemotherapy experiments combine this factor with thenegative influence of the cancer disease progress on the body weight andthe eventual beneficial therapeutic activity realized with the drugbeing used.

The control group in this study had a tendency to lose weight (evolutionfrom 106.3 to 101.6% of their initial value from day 6 to day 14). Inthe same period of time, groups treated with Compound II, DOX at 34.5μmol/kg and at 69.0 μmol/kg gained 4.9 and 4.1%, respectively. Thistendency observed in the control group may be attributed to theinfluence of the induced metastases on the experimental animals resultsconcerning the B16-B16 metastases in lungs of mice from this groupconfirm this conclusion with lungs severely affected (45.7±12.6% of thelung surface occupied with metastases).

Compound II, DOX gave excellent results in this experiment. Two groupsof mice having received 34.5 μmol/kg and at 69.0 μmol/kg, respectively,did not show any important weight losses during the experiment. Noclinical signs of toxicity were observed. At the same time, the drug hada marked effect on the metastatic growth. In the case of the dose of34.5 μmol/kg, the drug had a beneficial effect compared to the controlgroup with only 8.2±1.8% of the lung surface colonized (P<0.01). Thedrug given at 69.0 μmol/kg gave the value of 1.5%±0.6%, that is 15.6times lower than in the case of the highest DOX dose of 8.6 μmol/kg(P<0.001). DOX did not show any significant activity in either dosewith, however, some tendency to decrease the surface of lung metastasesat 8.6 μmol/kg (23.4±5.7% against 45.7±12.6% in the non-treated group).

EXAMPLE 8 Chemotherapeutic Activity of Compound II, DOX in the LS174THuman Colon Tumor Xenograft Model

The in vivo efficacy of Compound II, DOX by the i.p. route in micebearing LS174T colon tumor xenografts was studied. DOX was used as acontrol. These tumors are known to resist treatment with anthracyclines.Estimation of the antitumor activity of Compound II, DOX, whenadministered at equitoxic doses and in the same experimental model, isof crucial importance for validation of the prodrug and the concept ofan extracellularly tumor activated prodrug.

Female Swiss nude mice were 5 weeks old upon delivery (See GeneralMethods, Section O). They were kept under pathogen-free conditions withfood and water supplied ad libitum, and kept for about one week beforeimplantation of the tumors. Fragments (˜2 mm) of LS174T tumors grown innude mice were implanted subcutaneously in both flanks of the mice usedin the study. Tumors were left to grow for 17 days. At that time, themice weighed about 20.5-26.5 g.

Mice were then selected and assigned to groups in order to have equallydistributed tumor volumes in different groups (5-6 mice, 9-10tumors/group). The dose levels of Compound II, DOX used in the study,were selected on the basis of a lethality study previously conducted onOF-1 normal mice. The dose of DOX was based on previous chemotherapyexperiments performed in nude mice with the same dosing schedule.

Treatments were administered by the i.p. route on days 0, 1, 2, 3 and 4,the mice receiving a constant volume (10 μl/g) of either saline or thedifferent dosing solutions. DOX was used at 3.8 μmol/kg, Compound II,DOX was used at 25.9 μmol/kg and 34.5 μmol/kg. Clinical signs (recorded0.5, 1, 2 and 4 hours after dosing and daily between dosing days) andbody weights (recorded on each day of tumor measurement) were used toassess treatment toxicity, tumor growth was monitored on days 7, 12, 15,19, 22, 26, 29, 33 and 36 by two-dimensional measurements using calipersand a precision of 0.5 mm.

No treatment-related mortality and no significant weight loss wasobserved in the control and DOX-treated groups, as well as in the groupsthat received the lower dosage of Compound II, DOX.

In the Compound II, DOX at 34.5 μmol/kg group, three mice died beforethe end of the study, on days 18, 34 and 35, respectively. The mean bodyweight in this group reached its maximum of 14% of loss on day 26.According to the Mann-Whitney test, Compound II, DOX is the onlytreatment that resulted in significant differences as compared to thecontrol animals. Significant difference existed between the groupstreated with this compound and the DOX-treated group.

The plasmin-targeted prodrug clearly showed activity, the mostinteresting results being obtained in Compound II, DOX at 34.5 μmol/kgwith a minimal T/C ratio of 23% reached on day 33 and a specific growthdelay (one doubling of tumor size) of 1.7. However, this dose wasslightly toxic and higher than its maximum tolerated dose. It could beclearly seen however, that a therapeutic effect was observed with the25.9 μmol/kg dosage. Indeed, at 22 days, a significant median RTV valuereduction of 50% was observed. No tumor regression was observed in thisstudy in any of the DOX treatment groups. Considering the total lack ofactivity of DOX in the same model, these results are promising.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particularsituation, material, composition of matter, process, process step orsteps, while remaining within the scope of the present invention.Accordingly, the scope of the invention should therefore be determinedwith reference to the appended claims, along with the full range ofequivalents to which those claims are entitled.

1. A pharmaceutical composition comprising: (1) A compound comprising:(a) a therapeutic agent capable of entering a target tumor cell, whereinsaid therapeutic agent is daunorubicin or doxorubicin and plasmin ispresent in the extracellular vicinity of the target tumor cell; (b) anoligopeptide having the formula (AA^(x))_(m)-(AA^(y))_(n) (SEQ ID NO:1)wherein: (AA^(x))_(m) is a plasmin substrate and each AA^(x)independently represents an amino acid; each AA^(y) independentlyrepresents an amino acid selected from the group consisting of Ile, Leu,Phe, and Val; m is an integer from 2-4; and n is an integer from 1-2;(c) a stabilizing group; and (d) optionally, a linker group notcleavable by plasmin; wherein the oligopeptide is directly linked to thestabilizing group at a first attachment site of the oligopeptide and theoligopeptide is directly linked to the therapeutic agent or indirectlylinked through the linker group to the therapeutic agent at a secondattachment site of the oligopeptide; wherein the stabilizing grouphinders cleavage of the oligopeptide by enzymes present in whole blood;and wherein the compound is cleaved by plasmin under physiologicalconditions; and (2) a pharmaceutically acceptable carrier.
 2. A compoundcomprising: (1) a therapeutic agent capable of entering a target tumorcell, wherein said therapeutic agent is daunorubicin or doxorubicin andplasmin is present in the extracellular vicinity of the target tumorcell; (2) an oligopeptide having the formula X-Y, where X is a plasminpeptide substrate of 2-4 amino acids and Y is a peptide comprising 1-2amino acids each independently selected from the group consisting ofIle, Leu, Phe, and Val; (3) a stabilizing group; and (4) optionally, alinker group not cleavable by plasmin; wherein the oligopeptide isdirectly linked to the stabilizing group at a first attachment site ofthe oligopeptide and the oligopeptide is directly linked to thetherapeutic agent or indirectly linked through the linker group to thetherapeutic agent at a second attachment site of the oligopeptide;wherein the stabilizing group hinders cleavage of the oligopeptide byenzymes present in whole blood; and wherein the compound is cleavable byplasmin.
 3. The compound of claim 2 wherein plasmin cleaves the linkagebetween X and Y of the oligopeptide.
 4. The compound of claim 2 being aprodrug having an active portion, wherein the active portion of theprodrug is more capable of entering the target cell after cleavage bythe plasmin than prior to cleavage by plasmin, the active portionincluding at least the therapeutic agent.
 5. The compound of claim 4wherein the active portion of the prodrug consists of the therapeuticagent.
 6. The compound of claim 4 wherein the active portion of theprodrug includes the therapeutic agent and at least the linker group. 7.The compound of claim 4 wherein the active portion of the prodrugincludes the therapeutic agent and one amino acid of the Y portion ofthe oligopeptide.
 8. The compound of claim 7 further comprising twoamino acids of the Y portion of the oligopeptide.
 9. The compound ofclaim 2 wherein the oligopeptide is selected from: Leu-Lys-Leu- andLeu-Lys-Leu-Leu (SEQ ID NO:4).
 10. The compound of claim 2 wherein X is(AA^(x)), and Y is (AA^(y))_(m) wherein: each AA^(x) independentlyrepresents an amino acid; each AA^(y) independently represents an aminoacid; m is an integer from 2-4; n is an integer from 1-2; and AA^(x4) isan amino acid residue selected from the group consisting of Ile and Phe.11. The compound of claim 2 wherein X is (AA^(x)), and Y is (AA^(y))_(m)wherein: each AA^(x) independently represents an amino acid; each AA^(y)independently represents an amino acid; m is an integer from 2-4; n isan integer from 1-2; and AA^(x3) is an amino acid residue selected fromthe group consisting of Ala, Glu, Gly, Ile, Leu, Phe, Pro and Val. 12.The compound of claim 2 wherein X is (AA^(x)), and Y is (AA^(y))_(m)wherein: each AA^(x) independently represents an amino acid; each AA^(y)independently represents an amino acid; m is an integer from 2-4; n isan integer from 1-2; and AA^(x2) is an amino acid residue selected fromthe group consisting of Ala, Glu, Gly, Leu, Lys, Phe, Pro and Val. 13.The compound of claim 2 wherein X is (AA^(x)), and Y is (AA^(y))_(m)wherein: each AA^(x) independently represents an amino acid; each AA^(y)independently represents an amino acid; m is an integer from 2-4; n isan integer from 1-2; and AA^(x1) is an amino acid residue selected fromthe group consisting of Arg and Lys.
 14. The compound of claim 2 whereinX is (AA^(x)), and Y is (AA^(y))_(m)wherein: each AA^(x) independentlyrepresents an amino acid; each AA^(y) independently represents an aminoacid; m is an integer from 2-4; n is an integer from 1-2; and AA^(y2)and AA^(y1) are Leu residues.
 15. The compound of claim 2 wherein X is(AA^(x)), and Y is (AA^(y))_(m) wherein: each AA^(x) independentlyrepresents an amino acid; each AA^(y) independently represents an aminoacid; m is an integer from 2-4; n is an integer from 1-2; and AA^(y1) isa Leu residue and AA^(y2) is absent.
 16. The compound of claim 2 whereinthe stabilizing group is selected from the group consisting of D-Ala,P-Ala, .α-methyl-Ala, Succ-D-Ala, Succ-β-Ala and Succ-α-methyl-Ala. 17.The compound of claim 2 wherein the therapeutic agent is daunorubicin.18. The compound of claim 2 wherein the therapeutic agent isdoxorubicin.
 19. The compound of claim 2 wherein the therapeutic agenthas an intracellular active site.
 20. The compound of claim 2 whereinthe oligopeptide is directly linked to the therapeutic agent.
 21. Thecompound of claim 2 wherein the oligopeptide is indirectly linked to thetherapeutic agent at the second attachment site of the oligopeptide viaa linker group, and the linker group is selected from the groupconsisting of amino caproic acid, hydrazide group, an ester group, anether group, and a sulphydryl group.
 22. A method for treating a patientfor a tumor, the method comprising administering to the patient acompound comprising: (1) a therapeutic agent capable of entering atarget tumor cell, wherein said therapeutic agent is daunorubicin ordoxorubicin; (2) an oligopeptide having the formula(AA^(x))_(m)(AA^(y))_(n)(SEQ ID NO:1) wherein: (AA^(x))_(n) is a plasminsubstrate and each AA^(x) independently represents an amino acid; eachAA^(y) independently represents an amino acid selected from the groupconsisting of Ile, Leu, Phe, and Val; m is an integer from 2-4; and n isan integer from 1-2; (3) a stabilizing group; and (4) optionally, alinker group not cleavable by plasmin; wherein the oligopeptide isdirectly linked to the stabilizing group at a first attachment site ofthe oligopeptide and the oligopeptide is directly linked to thetherapeutic agent or indirectly linked through the linker group to thetherapeutic agent at a second attachment site of the oligopeptide;wherein the stabilizing group hinders cleavage of the oligopeptide byenzymes present in whole blood; wherein plasmin is present in theextracellular vicinity of the target tumor cell and wherein the compoundis cleaved by plasmin under physiological conditions.
 23. The method ofclaim 22 wherein the compound is administered intravenously.
 24. Themethod of claim 22 wherein the patient is treated for a medicalcondition selected from the group consisting of cancer, neoplasticdiseases, or tumors.